Microarray based multiplex pathogen analysis and uses thereof

ABSTRACT

Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5′ and 3′ ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation under 35 U.S.C. § 120 of pendingapplication U.S. Ser. No. 15/916,062, filed Mar. 8, 2018, which is acontinuation-In-part under 35 U.S.C. § 120 of pending non-provisionalapplication U.S. Ser. No. 15/388,561, filed Dec. 22, 2016, which claimsbenefit of priority under 35 U.S.C. § 119(e) of provisional applicationU.S. Ser. No. 62/271,371, filed Dec. 28, 2015, now abandoned, all ofwhich are hereby incorporated in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure is in the technical field of DNA based pathogenand plant analysis. More particularly, the present disclosure is in thetechnical field of pathogen analysis for plant, agriculture, food andwater material using a multiplex assay and a 3-dimensional latticemicroarray technology for immobilizing nucleic acid probes.

Description of the Related Art

Current techniques used to identify microbial pathogens rely uponestablished clinical microbiology monitoring. Pathogen identification isconducted using standard culture and susceptibility tests. These testsrequire a substantial investment of time, effort, cost as well as labileproducts. Current techniques are not ideal for testing large numberssamples. Culture-based testing is fraught with inaccuracies whichinclude both false positives and false negatives, as well as unreliablequantification of colony forming units (CFUs). There are issues with thepresence of viable but non-culturable microorganisms which do not showup using conventional culture methods. Certain culture tests are verynon-specific in terms of detecting both harmful and harmless specieswhich diminishes the utility of the test to determine if there is athreat present in the sample being tested.

In response to challenges including false positives and culturing ofmicroorganisms, DNA-based diagnostic methods such as polymerase chainreaction (PCR) amplification techniques were developed. To analyze apathogen using PCR, DNA is extracted from a material prior to analysis,which is a time-consuming and costly step.

In an attempt to eliminate the pre-analysis extraction step of PCR,Colony PCR was developed. Using cells directly from colonies from platesor liquid cultures, Colony PCR allows PCR of bacterial cells withoutsample preparation. This technique was a partial success but was not assensitive as culture indicating a possible issue with interference ofthe PCR by constituents in the specimens. Although this possibleinterference may not be significant enough to invalidate the utility ofthe testing performed, such interference can be significant for highlysensitive detection of pathogens for certain types of tests.Consequently, Colony PCR did not eliminate the pre-analysis extractionstep for use of PCR, especially for highly sensitive detection ofpathogens.

It is known that 16S DNA in bacteria and the ITS2 DNA in yeast or moldcan be PCR amplified, and once amplified can be analyzed to provideinformation about the specific bacteria or specific mold or yeastcontamination in or on plant material. Further, for certain samples suchas blood, fecal matter and others, PCR may be performed on the DNA insuch samples absent any extraction of the DNA. However, for blood it isknown that the result of such direct PCR is prone to substantial sampleto sample variation due to inhibition by blood analytes. Additionally,attempts to perform direct PCR analysis on plant matter have generallybeen unsuccessful, due to heavy inhibition of PCR by plant constituents.

Over time, additional methods and techniques were developed to improveon the challenges of timely and specific detection and identification ofpathogens. Immuno-assay techniques provide specific analysis. However,the technique is costly in the use of chemical consumables and has along response time. Optical sensor technologies produce fast real-timedetection but such sensor lack identification specificity as they offera generic detection capability as the pathogen is usually opticallysimilar to its benign background. Quantitative Polymerase Chain Reaction(qPCR) technique is capable of amplification and detection of a DNAsample in less than an hour. However, qPCR is largely limited to theanalysis of a single pathogen. Consequently, if many pathogens are to beanalyzed concurrently, as is the case with plant, agriculture, food andwater material, a relatively large number of individual tests areperformed in parallel.

Biological microarrays have become a key mechanism in a wide range oftools used to detect and analyze DNA. Microarray-based detectioncombines DNA amplification with the broad screening capability ofmicroarray technology. This results in a specific detection and improvedrate of process. DNA microarrays can be fabricated with the capacity tointerrogate, by hybridization, certain segments of the DNA in bacteriaand eukaryotic cells such as yeast and mold. However, processing a largenumber of PCR reactions for downstream microarray applications is costlyand requires highly skilled individuals with complex organizationalsupport. Because of these challenges, microarray techniques have not ledto the development of downstream applications.

It is well known that DNA may be linked to a solid support for thepurposes of DNA analysis. In those instances, the surface-associated DNAis generally referred to as the “Oligonucleotide probe” (nucleic acidprobe, DNA probe) and its cognate partner to which the probe is designedto bind is referred to as the Hybridization Target (DNA Target). In sucha device, detection and-or quantitation of the DNA Target is obtained byobserving the binding of the Target to the surface bound Probe viaduplex formation, a process also called “DNA Hybridization”(Hybridization).

Nucleic acid probe linkage to the solid support may be achieved bynon-covalent adsorption of the DNA directly to a surface as occurs whena nucleic acid probe adsorbs to a neutral surface such as cellulose orwhen a nucleic acid probe adsorbs to cationic surface such asamino-silane coated glass or plastic. Direct, non-covalent adsorption ofnucleic acid probes to the support has several limitations. The nucleicacid probe is necessarily placed in direct physical contact with thesurface thereby presenting steric constraints to its binding to a DNATarget as the desired (bound) Target-Probe complex is a double helixwhich can only form by wrapping of the Target DNA strand about the boundProbe DNA: an interaction which is fundamentally inhibited by directphysical adsorption of the nucleic acid probe upon the underlyingsurface.

Nucleic acid probe linkage may also occur via covalent attachment of thenucleic acid probe to a surface. This can be induced by introduction ofa reactive group (such as a primary amine) into the Probe then covalentattachment of the Probe, through the amine, to an amine-reactive moietyplaced upon the surface: such as an epoxy group, or an isocyanate group,to form a secondary amine or a urea linkage, respectively. Since DNA isnot generally reactive with epoxides or isocyanates or other similarstandard nucleophilic substitutions, the DNA Probe must be firstchemically modified with “unnatural” ligands such as primary amines orthiols. While such chemistry may be readily implemented duringoligonucleotide synthesis, it raises the cost of the DNA Probe by morethan a factor of two, due to the cost associated with the modificationchemistry. A related UV crosslinking based approach circumvents the needfor unnatural base chemistry, wherein Probe DNA can be linked to thesurface via direct UV crosslinking of the DNA, mediated by photochemicaladdition of thymine within the Probe DNA to the amine surface to form asecondary amine adduct. However, the need for high energy UV forefficient crosslinking results in substantial side reactions that candamage the nucleic acid probe beyond use. As is the case for adsorptivelinkage, the covalent linkages possible between a modified nucleic acidprobe and a reactive surface are very short, in the order of less than10 rotatable bonds, thereby placing the nucleic acid probe within 2 nmof the underlying surface. Given that a standard nucleic acid probeis >20 bases in length (>10 nm long) a Probe/linker length ratio >10/1also provides for destabilizing inhibition of the subsequent formationof the desired Target-Probe Duplex.

Previous Attempts at addressing these problems have not met withsuccess. Attachment of nucleic acid probes to surfaces via theirentrapment into a 3-Dimensional gel phase such as that created bypolymerizing acrylamide and polysaccharides among others have beenproblematic due to the dense nature of the gel phases. While the poresize (about 10 nm) in these gels permit entrapment and/or attachment ofthe nucleic acid probes within the gel, the solution-phase DNA Target,which is typically many times longer than the nucleic acid probe, isblocked from penetrating the gel matrix thereby limiting use of thesegel phase systems due to poor solution-phase access to the Target DNA.

Thus, the prior art is deficient in methods of DNA based pathogenanalysis that interrogates a multiplicity of samples, uses fewerchemical and labile products, reduces processing steps and providesfaster results while maintaining accuracy, specificity and reliability.The present invention fulfills this long-standing need and desire in theart.

SUMMARY OF THE INVENTION

The present invention is directed to a covalent 3-dimensional latticemicroarray system for DNA detection and analysis. The system comprises asolid support having chemically activatable groups on its surface, aplurality of bifunctional polymer linkers covalently attached to thesolid support and a plurality of nucleic acid probes covalently attachedto the bifunctional polymer linkers.

The present invention is directed to an adsorptive 3-dimensional latticemicroarray system for DNA detection and analysis. The system comprises asolid support having no chemically activatable groups on its surface, aplurality of bifunctional polymer linkers attached to the solid supportby non-covalent adsorption and a plurality of nucleic acid probesattached covalently to the bifunctional polymer linkers.

The present invention is also directed to a method for fabricating a3-dimensional lattice microarray system. The method comprises the stepsof contacting the solid support with a formulation comprising aplurality of nucleic acid probes, a plurality of fluorescentbifunctional polymer linkers and a solvent mixture comprising water anda boiling point water-miscible liquid; performing a first attachment toattach the fluorescent bifunctional polymer linker to the solid support,evaporating the water in the sample and performing a second attachmentstep to covalently attach the nucleic acid probes to the fluorescentbifunctional polymer linker.

The present invention is further directed to a customizable kitcomprising the solid support, a plurality of fluorescent labeledbifunctional polymer linkers, solvents and instructions for fabricatingthe microarray using a plurality of custom designed nucleic acid probesrelevant to an end user.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the embodiments ofthe present disclosure will become better understood when the followingdetailed description is read with reference to the accompanying drawingsin which like characters represent like parts throughout the drawing,wherein:

FIGS. 1A-1D illustrate a covalent microarray system comprising probesand bifunctional labels printed on an activated surface. FIG. 1A showsthe components—unmodified nucleic acid probe, amine-functionalized (NH)bifunctional polymer linker and amine-functionalized (NH) fluorescentlylabeled bifunctional polymer linker in a solvent comprising water and ahigh boiling point water-miscible liquid, and a solid support withchemically activatable groups (X). FIG. 1B shows the first step reactionof the bifunctional polymer linker with the chemically activated solidsupport where the bifunctional polymer linker becomes covalentlyattached by the amine groups to the chemically activated groups on thesolid support. FIG. 1C shows the second step of concentration viaevaporation of water from the solvent to increase the concentration ofthe reactants—nucleic acid probes and bifunctional polymer linker. FIG.1D shows the third step of UV crosslinking of the nucleic acid probesvia thymidine base to the bifunctional polymer linker within evaporatedsurface, which in some instances also serves to covalently link adjacentbifunctional polymeric linkers together via crosslinking to the nucleicacid Probe.

FIGS. 2A-2D illustrate an adsorptive microarray system comprising probesand bifunctional polymeric linkers. FIG. 2A shows the components;unmodified nucleic acid probe and functionalized (R_(n)) bifunctionalpolymer linker and similarly functionalized fluorescent labeledbifunctional polymer linker in a solvent comprising water and a highboiling point water-miscible liquid, and a solid support, wherein theR_(n) group is compatible for adsorbing to the solid support surface.FIG. 2B shows the first step adsorption of the bifunctional polymerlinker on the solid support where the bifunctional polymer linkersbecome non-covalently attached by the R_(n) groups to the solid support.FIG. 2C shows the second step of concentration via evaporation of waterfrom the solvent to increase the concentration of the reactants—Nucleicacid probes and bifunctional polymer linker. FIG. 2D shows the thirdstep of UV crosslinking of the nucleic acid probes via thymidine base tothe bifunctional polymer linker and other nucleic acid probes within theevaporated surface which in some instances also serves to covalentlylink adjacent bifunctional polymeric linkers together via crosslinkingto the nucleic acid Probe.

FIGS. 3A-3C show experimental data using the covalent microarray system.In this example of the invention the bifunctional polymeric linker was achemically modified 40 base long oligo deoxythymidine (OligodT) having aCy5 fluorescent dye attached at its 5′ terminus and an amino groupattached at its 3′ terminus, suitable for covalent linkage with aborosilicate glass solid support which had been chemically activated onits surface with epoxysilane. The nucleic acid probes comprisedunmodified DNA oligonucleotides, suitable to bind to the solution statetarget, each oligonucleotide terminated with about 5 to 7 thymidines, toallow for photochemical crosslinking with the thymidines in the topdomain of the polymeric (oligodT) linker.

FIG. 3A shows an imaged microarray after hybridization and washing, asvisualized at 635 nm. The 635 nm image is derived from signals from the(red) CY5 fluor attached to the 5′ terminus of the bifunctional polymerlinker (OligodT) which had been introduced during microarray fabricationas a positional marker in each microarray spot.

FIG. 3B shows a microarray imaged after hybridization and washing asvisualized at 532 nm. The 532 nm image is derived from signals from the(green) CY3 fluor attached to the 5′ terminus of PCR amplified DNAobtained during PCR Reaction #2 of a DNA containing sample.

FIG. 3C shows an imaged microarray after hybridization and washing asvisualized with both the 532 nm and 635 nm images superimposed. Thesuperimposed images display the utility of parallel attachment of aCy5-labelled OligodT positional marker relative to the sequence specificbinding of the CY3-labelled PCR product.

FIG. 4A is a graphical representation of the position of PCR primersemployed within the 16S locus (all bacteria) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 4B is a graphical representation of the position of PCR primersemployed within the stx1 locus (pathogenic E. coli) to be used to PCRamplify unpurified bacterial contamination obtained from Cannabis washand related plant wash. These PCR primers are used to amplify and dyelabel DNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 5A is a graphical representation of the position of PCR primersemployed as a two stage PCR reaction within the stx2 locus (pathogenicE. coli) to be used to PCR amplify unpurified bacterial contaminationobtained from Cannabis wash and related plant wash. These PCR primersare used to amplify and dye label DNA from such samples for bacterialanalysis via microarray hybridization.

FIG. 5B is a graphical representation of the position of PCR primersemployed within the invA locus (Salmonella) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 6 is a graphical representation of the position of PCR primersemployed within the tuf locus (E. coli) to be used to PCR amplifyunpurified bacterial contamination obtained from Cannabis wash andrelated plant wash. These PCR primers are used to amplify and dye labelDNA from such samples for bacterial analysis via microarrayhybridization.

FIG. 7 is a graphical representation of the position of PCR primersemployed within the ITS2 locus (yeast and mold) to be used to PCRamplify unpurified yeast, mold and fungal contamination obtained fromCannabis wash and related plant wash. These PCR primers are used toamplify and dye label DNA from such samples for yeast and mold analysisvia microarray hybridization.

FIG. 8 is a graphical representation of the position of PCR primersemployed within the ITS1 locus (Cannabis Plant Control) to be used toPCR amplify unpurified DNA obtained from Cannabis wash. These PCRprimers are used to amplify and dye label DNA from such samples for DNAanalysis via microarray hybridization. This PCR reaction is used togenerate an internal plant host control signal, via hybridization, to beused to normalize bacterial, yeast, mold and fungal signals obtained bymicroarray analysis on the same microarray.

FIG. 9 is a flow diagram illustrating the processing of unpurifiedCannabis wash or other surface sampling from Cannabis (and related plantmaterial) so as to PCR amplify the raw Cannabis or related plantmaterial, and then to perform microarray analysis on that material so asto analyze the pathogen complement of those plant samples

FIG. 10 is a representative image of the microarray format used toimplement the nucleic acid probes. This representative format comprises12 microarrays printed on a glass slide, each separated by a Teflondivider (left). Each microarray queries the full pathogen detectionpanel in quadruplicate. Also, shown is a blow-up (right) of one suchmicroarray for the analysis of pathogens in Cannabis and related plantmaterials. The Teflon border about each microarray is fit to localizeabout 50 μL fluid sample for hybridization analysis where fluorescentlabeled amplicons and placed onto the microarray for 30 min at roomtemperature, followed by washing at room temperature then microarrayimage scanning of the dye-labelled pathogen and host Cannabis DNA.

FIGS. 11A-11B shows representative microarray hybridization dataobtained from purified bacterial DNA standards (FIG. 11A) and purifiedfungal DNA standards (FIG. 11B). In each case, the purified bacterialDNA is PCR amplified as though it were an unpurified DNA, thenhybridized on the microarray via the microarray probes described above.The data show that in this microarray format, each of the bacteria canbe specifically identified via room temperature hybridization andwashing. Similarly, the purified fungal DNA is PCR amplified as thoughit were an unpurified DNA, then hybridized on the microarray via themicroarray probes described above. The data show that in this microarrayformat, each of the fungal DNAs can be specifically identified via roomtemperature hybridization and washing.

FIG. 12 shows representative microarray hybridization data obtained from5 representative raw Cannabis wash samples. In each case, the rawpathogen complement of these 5 samples is PCR amplified, then hybridizedon the microarray via the microarray probes described above. The datashow that in this microarray format, specific bacterial, yeast, mold andfungal contaminants can be specifically identified via room temperaturehybridization and washing.

FIG. 13 shows representative microarray hybridization data obtained froma representative raw Cannabis wash sample compared to a representative(raw) highly characterized, candida samples. In each case, the rawpathogen complement of each sample is PCR amplified, then hybridized onthe microarray via the microarray probes described above. The data showthat in this microarray format, specific fungal contaminants can bespecifically identified via room temperature hybridization and washingon either raw Cannabis wash or cloned fungal sample.

FIG. 14 shows a graphical representation of the position of PCR primersemployed in a variation of an embodiment for low level detection ofBacteria in the Family Enterobacteriaceae including E. coli. These PCRprimers are used to selectively amplify and dye label DNA from targetedorganisms for analysis via microarray hybridization.

FIG. 15A is a graphical representation of microarray hybridization datademonstrating low level detection of E. coli O157:H7 from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 15B is a graphical representation of microarray hybridization datademonstrating low level detection of E. coli O1111 from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 15C is a graphical representation of microarray hybridization datademonstrating low level detection of Salmonella enterica from certifiedreference material consisting of enumerated colonies of specifiedbacteria spiked onto Humulus lupulus, (Hop plant).

FIG. 16 shows diagrams for sample collection and preparation from twomethods. Both the tape pull and wash method are used to process samplesto provide a solution for microbial detection via microarray analysis.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of this invention, there is provided a 3-dimensionallattice microarray system for screening a sample for the presence of amultiplicity of DNA. The system comprises a chemically activatable solidsupport, a bifunctional polymer linker and a plurality of nucleic acidprobes designed to identify sequence determinants in plant, animal orpathogen DNA.

In this embodiment, the solid support may be made of any suitablematerial known in the art including but not limited to borosilicateglass, a thermoplastic acrylic resin such aspoly(methylmethacrylate-VSUVT (PMMA-VSUVT), a cycloolefin polymers suchas ZEONOR® 1060R, metals including, but not limited to gold andplatinum, plastics including, but not limited to polyethyleneterephthalate, polycarbonate, nylon, ceramics including, but not limitedto TiO₂, and Indium tin oxide (ITO) and engineered carbon surfacesincluding, but not limited to graphene. The solid support has a frontsurface and a back surface and may be activated on the front surfacewith suitable chemicals which include but are not limited toepoxysilane, isocyanate, succinimide, carbodiimide, aldehyde andmaleimide. These are well known in the art and one of ordinary skill inthis art would be able to readily functionalize any of these supports asdesired. In a preferred embodiment, the solid support is epoxysilanefunctionalized borosilicate glass support.

In this embodiment, the bifunctional polymer linker has a top domain anda bottom end. On the bottom end is attached a first reactive moiety thatallows covalent attachment to the chemically activatable groups in thesolid support. Examples of first reactive moieties include but are notlimited to an amine group, a thiol group and an aldehyde group.Preferably, the first reactive moiety is an amine group. On the topdomain of the bifunctional polymer linker is provided a second reactivemoiety that allows covalent attachment to the oligonucleotide probe.Examples of second reactive moieties include but are not limited tonucleotide bases like thymidine, adenine, guanine, cytidine, uracil andbromodeoxyuridine and amino acid like cysteine, phenylalanine, tyrosineglycine, serine, tryptophan, cystine, methionine, histidine, arginineand lysine. The bifunctional polymer linker may be an oligonucleotidesuch as OligodT, an amino polysaccharide such as chitosan, a polyaminesuch as spermine, spermidine, cadaverine and putrescine, a polyaminoacid, with a lysine or histidine, or any other polymeric compounds withdual functional groups which can be attached to the chemicallyactivatable solid support on the bottom end, and the nucleic acid probeson the top domain. Preferably, the bifunctional polymer linker isOligodT having an amine group at the 5′ end.

In this embodiment, the bifunctional polymer linker may be unmodified.Alternatively, the bifunctional polymer linker has a color orfluorescent label attached covalently. Examples of fluorescent labelsinclude, but are not limited to a Cy5, a DYLIGHT™ DY647, a ALEXA FLUOR®647, a Cy3, a DYLIGHT™ DY547, or a ALEXA FLUOR® 550. These may beattached to any reactive group including but not limited to, amine,thiol, aldehyde, sugar amido and carboxy on the bifunctional polymerlinker. The chemistries of such reactive groups are well known in theart and one or ordinary skill can readily identify a suitable group on aselected bifunctional polymer linker for attaching the fluorescentlabel. Preferably, the bifunctional polymer linker is Cy5-labeledOligodT having an amino group attached at its 3′terminus for covalentattachment to an activated surface on the solid support.

Also in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light.

In this embodiment, the fluorescent label (fluorescent tag) attached tothe bifunctional polymer linker is beneficial since it allows the userto image and detect the position of the individual nucleic acid probes(“spot”) printed on the microarray. By using two different fluorescentlabels, one for the bifunctional polymer linker and the second for theamplicons generated from the DNA being queried, the user can obtain asuperimposed image that allows parallel detection of those nucleic acidprobes which have been hybridized with amplicons. This is advantageoussince it helps in identifying the plant or pathogen comprised in thesample using suitable computer and software, assisted by a databasecorrelating nucleic acid probe sequence and microarray location of thissequence with a known DNA signature in plants, animals or pathogens. Anyemitter/acceptor fluorescent label pairs known in the art may be used.For example, the bifunctional polymer linker may be labeled withemitters such as a Cy5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while theamplicons may be labeled with acceptors such as Cy3, DYLIGHT™ DY547, orALEXA FLUOR® 550. Preferably, the emitter is Cy5 and the acceptor isCy3.

In another embodiment of this invention, there is provided a3-dimensional lattice microarray system for screening a sample for thepresence of a multiplicity of DNA. The system comprises a solid support,a fluorescent labeled bifunctional polymer linker and a plurality ofnucleic acid probes designed to identify sequence determinants in plant,animal or pathogen DNA.

In this embodiment, the solid support has a front surface and a backsurface. The front surface has non-covalent adsorptive properties forspecific functionalized group(s) present in the fluorescent labeledbifunctional polymer linker (described below). Examples of such solidsupport include, but are not limited to borosilicate glass, SiO2, metalsincluding, but not limited to gold and platinum, plastics including, butnot limited to polyethylene terephthalate, polycarbonate, nylon,ceramics including, but not limited to TiO₂, and Indium tin oxide (ITO)and engineered carbon surfaces including, but not limited to graphene.

In this embodiment, the fluorescent labeled bifunctional polymer linkerhas a top domain and a bottom end. On the bottom end is attached one ormore functional groups (designated by “R_(n)”) that are compatible fornon-covalent adsorptive attachment with the front surface of the solidsupport. Examples of compatible R groups include, but are not limitedto, single stranded nucleic acids (example, OligodT),amine-polysaccharide (example, chitosan), extended planar hydrophobicgroups (example, digoxigenin, pyrene, Cy-5 dye).

Further in this embodiment, on the top domain of the bifunctionalpolymer linker is provided a second reactive moiety that allows covalentattachment to the oligonucleotide probe. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. To the bottom endof the bifunctional polymer linker may be attached polymeric moleculesincluding, but not limited to an oligonucleotide such as OligodT, anamino polysaccharide such as chitosan, a polyamine such as spermine,spermidine, cadaverine and putrescine, a polyamino acid, with a lysineor histidine, or OligodT that is modified at its 5′ end with adigoxigenin, a pyrene or a Cy5 or any other polymeric molecules with orwithout chemical modification suitable for non-covalent attachment tothe solid support. On the top domain of these bifunctional polymerlinkers is attached, the nucleic acid probes. Preferably, thebifunctional polymer linker is OligodT.

In one aspect of this embodiment, the bifunctional polymer linker isunmodified. Alternatively, the bifunctional polymer linker may be afluorescent labeled bifunctional polymer linker. The fluorescent labelmay be, but is not limited to a Cy5, a DYLIGHT™ DY647, a ALEXA FLUOR®647, a Cy3, a DYLIGHT™ DY547, or a ALEXA FLUOR® 550 attached to anyreactive group including but not limited to, amine, thiol, aldehyde,sugar amido and carboxy on the bifunctional polymer linker. Thechemistries of such reactive groups are well known in the art and one orordinary skill can readily identify a suitable group on a selectedbifunctional polymer linker for attaching the fluorescent label.Preferably, the bifunctional polymer linker is Cy5-labeled OligodT.

Also in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light.

In this embodiment, the fluorescent label (fluorescent tag) attached tothe bifunctional polymer linker is beneficial since it allows the userto image and detect the position of the individual nucleic acid probes(“spot”) printed on the microarray. By using two different fluorescentlabels, one for the bifunctional polymer linker and the second for theamplicons generated from the DNA being queried, the user can obtain asuperimposed image that allows parallel detection of those nucleic acidprobes which have been hybridized with amplicons. This is advantageoussince it helps in identifying the plant or pathogen comprised in thesample using suitable computer and software, assisted by a databasecorrelating nucleic acid probe sequence and microarray location of thissequence with a known DNA signature in plants, animals or pathogens. Anyemitter/acceptor fluorescent label pairs known in the art may be used.For example, the bifunctional polymer linker may be labeled withemitters such as a Cy5, DYLIGHT™ DY647, or ALEXA FLUOR® 647, while theamplicons may be labeled with acceptors such as Cy3, DYLIGHT™ DY547, orALEXA FLUOR® 550. Preferably, the emitter is Cy5 and the acceptor isCy3.

In yet another embodiment of this invention, there is provided a methodfor fabricating a 3-dimensional lattice microarray system for thepurpose of screening a sample for the presence of a multiplicity of DNAin a sample. The method comprises, contacting a solid support with aformulation comprising a plurality of nucleic acid probes, a pluralityof fluorescent bifunctional polymer linkers and a solvent mixturecomprising water and a high boiling point, water-miscible liquid,allowing a first attachment between the fluorescent bifunctional polymerlinkers and the solid support to proceed, evaporating the water in thesolvent mixture thereby concentrating the nucleic acid probes andfluorescent labeled bifunctional polymer linkers, allowing a secondattachment between the nucleic acid probes and the fluorescentbifunctional polymer linker, and washing the solid support with at leastonce to remove unattached fluorescent bifunctional polymer linkers andnucleic acid probes.

In this embodiment, the contacting step is achieved by standard printingmethods known in the art including, but not limited to piezo-electricprinting, contact printing, ink jet printing and pipetting, which allowan uniform application of the formulation on the activated support. Forthis, any suitable solid support known in the art including but notlimited to borosilicate glass, a polycarbonate, a graphene, a gold, athermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT(PMMA-VSUVT) and a cycloolefin polymer such as ZEONOR® 1060R may beemployed.

In one aspect of this embodiment, the first attachment of thebifunctional polymer linker to the solid support is by non-covalentmeans such as by adsorption or electrostatic binding. In this case, thebifunctional polymer linkers with one or more functional groups(designated by “R_(n)”) on the bottom end, that are compatible forattachment with the front surface of the solid support will be used.Examples of compatible R groups include, but are not limited to, singlestranded nucleic acids (example, OligodT), amine-polysaccharide(example, chitosan), extended planar hydrophobic groups (example,digoxigenin, pyrene, Cy-5 dye). In another aspect of this embodiment,the first attachment of the bifunctional polymer linker to the solidsupport is by covalent coupling between chemically activatable groups onthe solid support and a first reactive moiety on the bottom end of thebifunctional polymer linker. Suitable chemicals including but are notlimited to epoxysilane, isocyanate, succinimide, carbodiimide, aldehydeand maleimide may be used for activating the support. These are wellknown in the art and one of ordinary skill in this art would be able toreadily functionalize any of these supports as desired. In a preferredembodiment, a borosilicate glass support that is epoxysilanefunctionalized is used. Examples of first reactive moieties amenable tocovalent first attachment include, but are not limited to an aminegroup, a thiol group and an aldehyde group. Preferably, the firstreactive moiety is an amine group.

In this embodiment, the bifunctional polymer linker has a secondreactive moiety attached at the top domain. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. Preferably, thesecond reactive moiety is thymidine. In this aspect of the invention,the bifunctional polymer linker may be an oligonucleotide such asOligodT, an amino polysaccharide such as chitosan, a polyamine such asspermine, spermidine, cadaverine and putrescine, a polyamino acid, witha lysine or histidine, or any other polymeric compounds with dualfunctional groups which can be attached to the chemically activatablesolid support on the bottom end, and the nucleic acid probes on the topdomain. Preferably, the bifunctional polymer linker is OligodT having anamine group at the 5′ end.

In this embodiment, the bifunctional polymer linkers are modified with afluorescent label. Examples of fluorescent labels include but are notlimited Cy5, DYLIGHT™ DY647, ALEXA FLUOR® 647, Cy3, DYLIGHT™ DY547 andALEXA FLUOR® 550 attached to any reactive group including but notlimited to, amine, thiol, aldehyde, sugar amido and carboxy on thebifunctional polymer linker. The chemistries of such reactive groups arewell known in the art and one or ordinary skill can readily identify asuitable group on a selected bifunctional polymer linker for attachingthe fluorescent label. Preferably, the bifunctional polymer linker usedfor fabricating the microarray is Cy5-labeled OligodT.

The method of fabricating the microarray requires use of a solventmixture comprising water and a water-miscible liquid having a boilingpoint above 100° C. This liquid may be any suitable water-miscibleliquid with a boiling point higher than that of water, so that all thesolvent is not lost during the evaporation step. This allows themolecular reactants—nucleic acid probes and bifunctional linkers to beprogressively concentrated during evaporation. Such controlledevaporation is crucial to the present invention since it controls thevertical spacing between nucleic acid probes their avoiding sterichindrance during the hybridization steps thereby improving accuracy andprecision of the microarray. Examples of high boiling pointwater-miscible solvent include but are not limited to glycerol, DMSO andpropanediol. The ratio or water to high boiling point solvent is keptbetween 10:1 and 100:1 whereby, in the two extremes, upon equilibrium,volume of the fluid phase will reduce due to water evaporation tobetween 1/100th and 1/10^(th) of the original volume, thus giving riseto a 100-fold to 10-fold increase in reactant concentration. In apreferred embodiment, the water-miscible solvent is propanediol and thewater to propanediol ratio is 100:1.

Further in this embodiment, the nucleic acid probes used in the methodof microarray fabrication are designed with terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling during the fabrication process.Preferably, coupling of the nucleic acid probes to the fluorescentlabeled bifunctional polymer linkers is by photochemical covalentcrosslinking.

In yet another embodiment of this invention, there is provided acustomizable microarray kit. The kit comprises a solid support, aplurality of fluorescent labeled bifunctional polymer linkers, nucleicacid probes and a solvent mixture comprising water and one or more of awater-miscible liquid having a boiling point above 100° C., andinstructions to use the kit. Each of the components comprising this kitmay be individually customized prior to shipping based on the goals ofthe end user.

In this embodiment, the solid support has a front surface and a backsurface and made of any suitable material known in the art including butnot limited to borosilicate glass, a polycarbonate, a graphene, a gold,a thermoplastic acrylic resin such as poly(methylmethacrylate-VSUVT(PMMA-VSUVT) and a cycloolefin polymer such as sold under the trademarkZEON® 1060R owned by Zeon Chemicals.

In one aspect of this embodiment, the solid support is unmodified andhas properties capable of non-covalent attachment to groups in thebifunctional polymer linker. Alternatively, the solid support isactivated on the front surface with chemically activatable groups whichinclude but are not limited to epoxysilane, isocyanate, succinimide,carbodiimide, aldehyde and maleimide. These are well known in the artand one of ordinary skill in this art would be able to readilyfunctionalize any of these supports as desired. In a preferredembodiment, the solid support is epoxysilane functionalized borosilicateglass support.

In this embodiment, the bifunctional polymer linker has a top domain anda bottom end. In one aspect of this embodiment, to the bottom end of thebifunctional polymer linker are attached one or more functional groups(designated by “R_(n)”), which are compatible for attachment with thefront surface of the solid support in a non-covalent binding. Examplesof such compatible R groups include, but are not limited to, singlestranded nucleic acids (example, OligodT), amine-polysaccharide(example, chitosan), extended planar hydrophobic groups (example,digoxigenin, pyrene, Cy-5 dye). Alternatively, to the bottom end of thebifunctional polymer linker are attached a first reactive moiety thatallows covalent attachment to chemically activatable groups in the solidsupport. Examples of first reactive moieties include but are not limitedto an amine group, a thiol group and an aldehyde group. Preferably, thefirst reactive moiety is an amine group.

Further in this embodiment, on the top domain of the bifunctionalpolymer linker is provided a second reactive moiety that allows covalentattachment to the oligonucleotide probe. Examples of second reactivemoieties include but are not limited to nucleotide bases like thymidine,adenine, guanine, cytidine, uracil and bromodeoxyuridine and amino acidlike cysteine, phenylalanine, tyrosine glycine, serine, tryptophan,cystine, methionine, histidine, arginine and lysine. The bifunctionalpolymer linker may be an oligonucleotide such as OligodT, an aminopolysaccharide such as chitosan, a polyamine such as spermine,spermidine, cadaverine and putrescine, a polyamino acid, with a lysineor histidine, or any other polymeric compounds with dual functionalgroups for attachment to the solid support from the bottom end, and thenucleic acid probes from the top domain.

In one aspect of this embodiment, the bifunctional polymer linkers aremodified with a fluorescent label. Alternatively, the bifunctionalpolymer linker may be a fluorescent labeled bifunctional polymer linkerwhere the fluorescent label is a fluorescent cyanine dye Cy5 or Cy3, afluorescent phosphoramidite dye sold under the trademark DYLIGHT™ DY647or DYLIGHT™ DY547, or a fluorescent dye sold under the trademark ALEXAFLUOR® 647 all owned by Thermo Fisher Scientific Inc attached to anyreactive group including but not limited to, amine, thiol, aldehyde,sugar amido and carboxy on the bifunctional polymer linker. Thechemistries of such reactive groups are well known in the art and one orordinary skill can readily identify a suitable group on a selectedbifunctional polymer linker for attaching the fluorescent label.Preferably, the bifunctional polymer linker is Cy5-labeled OligodT.

Also in this embodiment, the present invention provides a plurality ofnucleic acid probes designed with the purpose of identifying sequencedeterminants in plants, animals or pathogens. The nucleic acid probesare synthetic oligonucleotides and have terminal thymidine bases attheir 5′ and 3′ end. The thymidine bases permit covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker by anystandard coupling procedures including but not limited to chemical,photochemical and thermal coupling. Preferably, covalent attachment ofthe nucleic acid probes to the bifunctional polymer linker is byphotochemical means using ultraviolet light.

In yet another embodiment of this invention there is provided a methodfor detecting the presence of one or more pathogens in a plant sample.In this embodiment, the pathogen may be a human pathogen, an animalpathogen or a plant pathogen, such as a bacterium, a fungus, a virus, ayeast, algae or a protozoan or a combination thereof. These pathogensmay be present as constituents of the soil, soilless growth media,hydroponic growth media or water in which the plant sample was grown.The method comprises harvesting the pathogens from the plant sample,isolating total nucleic acids comprising pathogen DNA, performing afirst amplification for generating one or more amplicons from the one ormore pathogens present in the sample in a single, simultaneous step;performing a labeling amplification using as template, the one or moreamplicons generated in the first amplification step to generatefluorescent labeled second amplicons; hybridizing the second ampliconswith the nucleic acid probes immobilized on the fabricatedself-assembled, 3-dimensional lattice microarray described above andimaging the microarray to detect the fluorescent signal, which indicatespresence of the one or more pathogens in a plant sample. In thisembodiment, the pathogens present on the plant surface may be harvestedby washing the plant with water to recover the pathogens, followed byconcentrating by filtration on a sterile 0.4 μm filter. In anotheraspect of this embodiment, pathogens within the plant tissue may beharvested by fluidizing the plant tissue sample and pathogens, followedby centrifuging to get a pellet of plant cells and pathogen cells. Ineither embodiment, the harvested sample is disrupted to release thetotal nucleic acids which is used in the subsequent steps withoutfurther purification.

Also in this embodiment, the sample comprising nucleic acids frompathogens (external pathogens) or nucleic acids from both pathogens andplant (internal pathogens) is used to perform a first amplification ofpathogen DNA using pathogen-specific first primer pairs to obtain one ormore pathogen-specific first amplicons. Any DNA amplificationmethodology, including loop-mediated isothermal amplification (LAMP) orpolymerase chain reaction (PCR) that can selectively amplify the DNAcomplement in the sample may be employed. In a preferred embodiment, theamplification is by PCR. In one embodiment, the pathogen is a bacteriumand the first primer pairs have sequences shown in SEQ ID NOS: 1-12. Inanother embodiment, the pathogen is a fungus and the first primer pairshave sequences shown in SEQ ID NOS: 13-16. An aliquot of first ampliconsso generated is used as template for a second, labelling PCRamplification using fluorescent labeled second primer pairs. The secondprimer pairs are designed to amplify an internal flanking region in theone or more first amplicons to obtain one or more first fluorescentlabeled second amplicons. In one embodiment, the pathogen is a bacteriumand the second primer pairs have sequences shown in SEQ ID NOS: 19-30.In another embodiment, the pathogen is a fungus and the second primerpairs have sequences shown in SEQ ID NOS: 31-34.

Further in this embodiment, the fluorescent labeled second amplicons arehybridized on a 3-dimensional lattice microarray system having aplurality of nucleic acid probes as described in detail above. In thisembodiment, the bifunctional polymer linker has a fluorescent label(that is different from the label on the second amplicon) attachedwhereby, imaging the microarray after hybridization and washing resultsin two distinct fluorescent signals—the signal from the fluorescentbifunctional polymer linker which is covalently linked to the nucleicacid probe during fabrication, which would be detected in each spotcomprised in the microarray, and a second amplicon signal that would bedetected only in those spots where the nucleic acid probe sequence iscomplementary to the second amplicon (originally derived byamplification from the pathogen DNA in the sample). Thus, superimposingthe two images using a computer provides beneficial attributes to thesystem and method claimed in this invention since one can readilyidentify the plant or pathogen comprised in the sample from a databasethat correlates nucleic acid probe sequence and microarray location ofthis sequence with a known DNA signature in plants or pathogens. In apreferred embodiment, the bacterial nucleic acid probes having sequencesshown in SEQ ID NOS: 37-85. and fungal nucleic acid probes havingsequences shown in SEQ ID NOS: 86-125 may be used for this purpose.

Further to this embodiment is a method for detecting plant DNA. Theplant may be a terrestrial plant such as a Humulus or a Cannabis, anaquatic plant, an epiphytic plant or a lithophytic plant that grows insoil, soilless media, hydroponic growth media or water. In a preferredaspect, the plant is a Cannabis. This method comprises the steps ofperforming an amplification on an unpurified complex nucleic acid sampleusing plant-specific first primer pairs to generate plant-specific firstamplicons. In one aspect of this embodiment, the first primer pair hassequences shown in SEQ ID NOS: 17-18. Any DNA amplification methodology,including loop-mediated isothermal amplification (LAMP) or polymerasechain reaction (PCR) that can selectively amplify the DNA complement inthe sample may be employed. Preferably the amplification is by PCR. Thefirst amplicons so generated are used as template for a labelingamplification step using fluorescent labeled second primer pairs thatare designed to amplify an internal flanking region in the one or moreof first amplicons generated in the first amplification step to generateone or more first fluorescent labeled second amplicons. In oneembodiment, the second primer pair has sequences shown in SEQ ID NOS:35-36. The second amplicons are hybridized on a 3-dimensional latticemicroarray system having a plurality of plant-specific nucleic acidprobes, and the microarrays imaged and analyzed as described above foridentifying pathogen DNA. In one aspect of this embodiment, thehybridization nucleic acid probes have sequences shown in SEQ ID NOS:126-128.

In yet another embodiment of this invention, there is provided a methodfor simultaneously detecting resident pathogen DNA and plant DNA in aplant sample in a single assay. In this embodiment, the pathogen may bea human pathogen, an animal pathogen or a plant pathogen, which may be abacterium, a fungus, a virus, a yeast, algae or a protozoan or acombination thereof. These pathogens may be present as constituents ofthe soil, soilless growth media, hydroponic growth media or water inwhich the plant sample was grown. The plant may be a terrestrial plantsuch as a Humulus or a Cannabis, an aquatic plant, an epiphytic plant ora lithophytic plant that grows in soil, soilless media, hydroponicgrowth media or water. Preferably, the plant is a Cannabis.

In this embodiment, the method comprises harvesting a plant tissuesample potentially comprising one or more pathogens, fluidizing theplant tissue sample and the one or more pathogens and isolating totalnucleic acids comprising DNA from at least the plant tissue and DNA fromthe one or more pathogens. In one aspect of this embodiment, the step ofisolating total nucleic acids comprises centrifuging the fluidizedsample to get a pellet of plant cells and pathogen cells which aredisrupted to release the total nucleic acids, which are used in thesubsequent steps without further purification.

Further in this embodiment, a first amplification is performed on theunpurified total nucleic acid sample using one or more of a first primerpair each selective for the one or more pathogen DNA and one or more ofa second primer pair selective for the plant DNA to generate one or morepathogen-specific first amplicons and one or more plant-specific secondamplicons. Any DNA amplification methodology, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) thatcan selectively amplify the DNA complement in the sample may beemployed. In a preferred embodiment, the amplification is by PCR. In oneembodiment, the pathogen is a bacterium and the first primer pairs havesequences shown in SEQ ID NOS: 1-12. In another embodiment, the pathogenis a fungus and the first primer pairs have sequences shown in SEQ IDNOS: 13-16. In either of these embodiments, the plant-specific secondprimer pairs have sequences shown in SEQ ID NOS: 35-36. An aliquot ofthe first and second amplicons so generated is used as a template for asecond, labeling PCR amplification step using fluorescent labeled thirdprimer pairs having a sequence complementary to an internal flankingregion in the one or more pathogen-specific first amplicons andfluorescent labeled fourth primer pairs having a sequence complementaryto an internal flanking region in the one or more plant-specific secondamplicons. Any DNA amplification methodology, including loop-mediatedisothermal amplification (LAMP) or polymerase chain reaction (PCR) thatcan selectively amplify the DNA complement in the sample may beemployed. In a preferred embodiment, the amplification is by PCR. In oneembodiment, the pathogen is a bacterium and the third primer pairs havesequences shown in SEQ ID NOS: 19-30. In another embodiment, thepathogen is a fungus and the third primer pairs have sequences shown inSEQ ID NOS: 31-34. In either of these embodiments, the plant-specificfourth primer pairs have sequences shown in SEQ ID NOS: 35-36. Thelabeling PCR step results in generation of first fluorescent labeledthird amplicons and second fluorescent labeled fourth ampliconscorresponding to the pathogen and plant DNA respectively in the originalharvested sample. These amplicons are then hybridized on a 3-dimensionallattice microarray system having a plurality of nucleic acid probesspecific to sequence determinants in pathogen DNA or plant DNA.Bacterial nucleic acid probes having sequences shown in SEQ ID NOS:37-85, fungal nucleic acid probes having sequences shown in SEQ ID NOS:86-125 and plant nucleic acid probes having sequences shown in SEQ IDNOS: 126-128. may be used for this purpose. After hybridization,unhybridized amplicons are removed by washing and the microarray imaged.Detection of the first fluorescent labeled third amplicon signalindicates presence of pathogens in the plant sample. Detecting thesecond fluorescent labeled fourth amplicon indicates presence of theplant DNA. Superimposing these two signals with the third fluorescentsignal from the fluorescent bifunctional polymer linker-coupled nucleicacid probes allow simultaneous identification of the pathogen and plantin the sample by correlating nucleic acid probe sequence and microarraylocation of this sequence with a known DNA signature in plants orpathogens. These features provide beneficial attributes to the systemand method claimed in this invention.

In yet another embodiment of the present disclosure there is provided animproved method for DNA based pathogen analysis. The embodiments of thepresent disclosure use DNA amplification methodologies, includingloop-mediated isothermal amplification (LAMP) or polymerase chainreaction (PCR) tests that can selectively amplify the DNA complement ofthat plant material using unpurified plant and pathogen material. Theembodiments are also based on the use of aforementioned PCR-amplifiedDNA as the substrate for microarray-based hybridization analysis,wherein the hybridization is made simple because the nucleic acid probesused to interrogate the DNA of such pathogens are optimized to functionat room temperature. This enables the use of the above-mentionedmicroarray test at ambient temperature, thus bypassing the prior artrequirement that testing be supported by an exogenoustemperature-regulating device.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1

Fabrication of 3-Dimensional Lattice Microarray Systems

The present invention teaches a way to link a nucleic acid probe to asolid support surface via the use of a bifunctional polymeric linker.The nucleic acid probe can be a PCR amplicon, syntheticoligonucleotides, isothermal amplification products, plasmids or genomicDNA fragment in a single stranded or double stranded form. The inventioncan be sub-divided into two classes, based on the nature of theunderlying surface to which the nucleic acid probe would be linked.

1. Covalent Microarray System with Activated Solid Support.

The covalent attachment of any one of these nucleic acid probes does notoccur to the underlying surface directly, but is instead mediatedthrough a relatively long, bi-functional polymeric linker that iscapable of both chemical reaction with the surface and also capable ofefficient UV-initiated crosslinking with the nucleic acid probe. Themechanics of this process is spontaneous 3D self assembly and isillustrated in FIG. 1A-FIG. 1D. As seen in FIG. 1A, the componentsrequired to fabricate this microarray system are:

(a) an unmodified nucleic acid probe 3 such as an oligonucleotide, PCRor isothermal amplicon, plasmid or genomic DNA;

(b) a chemically activatable surface 1 with chemically activatablegroups (designated “X”) compatible for reacting with a primary aminesuch as. epoxysilane, isocyanate, succinimide, carbodiimide, aldehyde.

(c) bifunctional polymer linkers 2 such as a natural or modifiedOligodT, amino polysaccharide, amino polypeptide suitable for couplingto chemically activatable groups on the support surface, each attachedwith a fluorescent label 4; and

(d) a solvent comprising water and a high boiling point, water-miscibleliquid such as glycerol, DMSO or propanediol (water to solvent ratiobetween 10:1 and 100:1).

Table 1 shows examples of chemically activatable groups and matchedreactive groups on the bifunctional polymer linker for mere illustrationpurposes only and does not in any way preclude use of other combinationsof matched reactive pairs.

TABLE 1 Covalent Attachment of Bifunctional Polymeric Linker to anActivated Surfaces Activated Surface Matched Reactive Group SpecificImplementation as Bifunctional Moiety on Bifunctional Linker polymericlinker Epoxysilane Primary Amine (1) Amine-modified OligodT (20-60bases) (2) Chitosan (20-60 subunits) (3) Lysine containing polypeptide(20-60aa) EDC Activated Primary Amine (4) Amine-modified OligodT (20-60bases) Carboxylic Acid (5) Chitosan (20-60 subunits) (6) Lysinecontaining polypeptide (20-60aa) N-hydroxysuccinimide Primary Amine (7)Amine-modified OligodT (20-60 bases) (NHS) (8) Chitosan (20-60 subunits)(9) Lysine containing polypeptide (20-60aa)

When used in the present invention, the chemically activatable surface,bifunctional polymer linkers and unmodified nucleic acid probes areincluded as a solution to be applied to a chemically activated surface 4by ordinary methods of fabrication used to generate DNA Hybridizationtests such as contact printing, piezo electric printing, ink jetprinting, or pipetting.

Microarray fabrication begins with application of a mixture of thechemically activatable surface, bifunctional polymer linkers andunmodified nucleic acid probes to the surface. The first step isreaction and covalent attachment of the bifunctional linker to theactivated surface (FIG. 1B). In general, the chemical concentration ofthe bi-functional linker is set to be such that less than 100% of thereactive sites on the surface form a covalent linkage to thebi-functional linker. At such low density, the average distance betweenbi-functional linker molecules defines a spacing denoted lattice width(“LW” in FIG. 1B).

In the second step, the water in the solvent is evaporated toconcentrate the DNA and bifunctional linker via evaporation of waterfrom the solvent (FIG. 1C). Generally, use of pure water as the solventduring matrix fabrication is disadvantageous because water is veryquickly removed by evaporation due to a high surface area/volume ratio.To overcome this, in the present invention, a mixture of water with ahigh boiling point water-miscible solvent such as glycerin, DMSO orpropanediol was used as solvent. In this case, upon evaporation, thewater component will evaporate but not the high boiling point solvent.As a result, molecular reactants—DNA and bifunctional linker areprogressively concentrated as the water is lost to evaporation. In thepresent invention, the ratio or water to high boiling point solvent iskept between 10:1 and 100:1. Thus, in the two extreme cases, uponequilibrium, volume of the fluid phase will reduce due to waterevaporation to between 1/100th and 1/10^(th) the original volume, thusgiving rise to a 100-fold to 10-fold increase in reactant concentration.Such controlled evaporation is crucial to the present invention since itcontrols the vertical spacing (Vertical Separation, “VG” in FIG. 1C)between nucleic acid probes, which is inversely related to the extent ofevaporative concentration.

In the third step, the terminal Thymidine bases in the nucleic acidprobes are UV crosslinked to the bifunctional linker within theevaporated surface (FIG. 1D). This process is mediated by the well-knownphotochemical reactivity of the Thymidine base that leads to theformation of covalent linkages to other thymidine bases in DNA orphotochemical reaction with proteins and carbohydrates. If thebifunctional crosslinker is OligodT, then the crosslinking reaction willbe bi-directional, that is, the photochemistry can be initiated ineither the nucleic acid probe or the bifunctional OligodT linker. On theother hand, if the bifunctional linker is an amino polysaccharide suchas chitosan or a polyamino acid, with a lysine or histidine in it, thenthe photochemistry will initiate in the nucleic acid probe, with thebifunctional linker being the target of the photochemistry.

2. Microarray System with Unmodified Solid Support for Non-CovalentAttachment

In this microarray system, attachment of the nucleic acid probes doesnot occur to the underlying surface directly, but is instead mediatedthrough a relatively long, bi-functional polymeric linker that bindsnon-covalently with the solid support, but covalently with the nucleicacid probes via UV-initiated crosslinking. The mechanics of this processis spontaneous 3D self assembly and is illustrated in FIGS. 2A-2D. Asseen in FIG. 2A, the components required to fabricate this microarraysystem are:

-   -   (1) an unmodified nucleic acid probe 3 such as an        oligonucleotide, PCR or isothermal amplicon, plasmid or genomic        DNA;    -   (2) an unmodified solid support 1    -   (3) bifunctional polymer linkers 2 such as OligodT or a amino        polysaccharide, amino polypeptide, that inherently have or are        modified to have functional groups (designated “R”) compatible        for adsorptive binding to the solid support, each having a        fluorescent label 4; and    -   (4) a solvent comprising water and a high boiling point,        water-miscible liquid such as glycerol, DMSO or propanediol        (water to solvent ratio between 10:1 and 100:1);

Table 2 shows examples of unmodified support surfaces and matchedabsorptive groups on the bifunctional polymer linker for mereillustration purposes only and does not in any way precludes the use ofother combinations of these.

TABLE 2 Non-Covalent Attachment of Bi-Functional Polymeric Linker to anInert Surface Representative Matched Adsorptive Group SpecificBifunctional support surface on Bifunctional Linker (R_(n)) polymericlinker glass Single Stranded Nucleic OligodT (30-60 bases) Acid > 10bases glass Amine-Polysaccharide Chitosan (30-60 subunits) glassExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′-Digoxigenin polycarbonate Single Stranded NucleicOligo-dT (30-60 bases) Acid > 10 bases polycarbonateAmine-Polysaccharide Chitosan (30-60 subunits) polycarbonate ExtendedPlanar Hydrophobic OligodT (30-60 bases)- Groups e.g. Digoxigenin5′-Digoxigenin graphene Extended Planar Hydrophobic OligodT (30-60bases)- Groups e.g. pyrene 5′pyrene graphene Extended Planar HydrophobicOligodT (30-60 bases)- Groups e.g. CY-5 dye 5′-CY-5 dye grapheneExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′-Digoxigenin gold Extended Planar Hydrophobic OligodT(30-60 bases)- Groups e.g. pyrene 5′pyrene gold Extended PlanarHydrophobic OligodT (30-60 bases)- Groups e.g. CY-5 dye 5′ CY-5 dye goldExtended Planar Hydrophobic OligodT (30-60 bases)- Groups e.g.Digoxigenin 5′ Digoxigenin

When used in the present invention, components 1-3 are included as asolution to be applied to the solid support surface by ordinary methodsof fabrication used to generate DNA Hybridization tests such as contactprinting, piezo electric printing, ink jet printing, or pipetting.

Microarray fabrication begins with application of a mixture of thecomponents (1)-(3) to the surface. The first step is adsorption of thebifunctional linker to the support surface (FIG. 2B). The concentrationof the bi-functional linker is set so the average distance betweenbi-functional linker molecules defines a spacing denoted as latticewidth (“LW” in FIG. 2B).

In the second step, the water in the solvent is evaporated toconcentrate the DNA and bifunctional linker via evaporation of waterfrom the solvent (FIG. 2C). Generally, use of pure water as the solventduring matrix fabrication is disadvantageous because water is veryquickly removed by evaporation due to a high surface area/volume ratio.To overcome this, in the present invention, a mixture of water with ahigh boiling point water-miscible solvent such as glycerin, DMSO orpropanediol was used as solvent. In this case, upon evaporation, thewater component will evaporate but not the high boiling point solvent.As a result, molecular reactants—DNA and bifunctional linker areprogressively concentrated as the water is lost to evaporation. In thepresent invention, the ratio or water to high boiling point solvent iskept between 10:1 and 100:1. Thus, in the two extreme cases, uponequilibrium, volume of the fluid phase will reduce due to waterevaporation to between 1/100th and 1/10^(th) the original volume, thusgiving rise to a 100-fold to 10-fold increase in reactant concentration.

In the third step, the terminal Thymidine bases in the nucleic acidprobes are UV crosslinked to the bifunctional linker within theevaporated surface (FIG. 2D). This process is mediated by the well-knownphotochemical reactivity of the Thymidine base that leads to theformation of covalent linkages to other thymidine bases in DNA orphotochemical reaction with proteins and carbohydrates. If thebifunctional crosslinker is OligodT, then the crosslinking reaction willbe bi-directional, that is, the photochemistry can be initiated ineither the nucleic acid probe or the bifunctional OligodT linker. On theother hand, if the bifunctional linker is an amino polysaccharide suchas chitosan or a polyamino acid, with a lysine or histidine in it, thenthe photochemistry will initiate in the nucleic acid probe, with thebifunctional linker being the target of the photochemistry.

Although such non-covalent adsorption described in the first step isgenerally weak and reversible, when occurring in isolation, in thepresent invention it is taught that if many such weak adsorptive eventsbetween the bifunctional polymeric linker and the underlying surfaceoccur in close proximity, and if the closely packed polymeric linkersare subsequently linked to each other via Thymidine-mediatedphotochemical crosslinking, the newly created extended, multi-molecular(crosslinked) complex will be additionally stabilized on the surface,thus creating a stable complex with the surface in the absence of directcovalent bonding to that surface.

The present invention works very efficiently for the linkage ofsynthetic oligonucleotides as nucleic acid probes to form amicroarray-based hybridization device for the analysis of microbial DNAtargets. However, it is clear that the same invention may be used tolink PCR amplicons, synthetic oligonucleotides, isothermal amplificationproducts, plasmid DNA or genomic DNA fragment as nucleic acid probes. Itis also clear that the same technology could be used to manufacturehybridization devices that are not microarrays.

DNA nucleic acid probes were formulated as described in Table 3, to bedeployed as described above and illustrated in FIG. 1 or 2. A set of 48such probes (Table 4) were designed to be specific for various sequencedeterminants of microbial DNA and each was fabricated so as to present astring of 5-7 T bases at each end, to facilitate their UV-crosslinkingto form a covalently linked microarray element, as described above andillustrated in FIG. 1. Each of the 48 different probes was printed intriplicate to form a 144 element (12×12) microarray having sequencesshown in Table 3.

TABLE 3 Representative Conditions of use of the Present Invention 5′labelled Unique sequence OligodT Oligonucleotide Fluorescent Nucleicacid probe 30-38 bases Long marker 30 bases Type 7 T's at each endLong(marker) Nucleic acid probe 50 mM 0.15 mM Concentration BifunctionalLinker OligodT 30 bases long Primary amine at 3′ terminus BifunctionalLinker 1 mM Concentration High Boiling point Water: Propanediol, Solvent100:1 Surface Epoxysilane on borosilicate glass UV Crosslinking Dose 300millijoule (mjoule)

TABLE 4Nucleic acid probes Linked to the Microarray Surface via the Present InventionSEQ ID NO: 132 Negative control TTTTTTCTACTACCTATGCTGATTCACTCTTTTTSEQ ID NO: 129 Imager Calibration TTTTCTATGTATCGATGTTGAGAAATTTTTTT(High) SEQ ID NO: 130 Imager Calibration (Low)TTTTCTAGATACTTGTGTAAGTGAATTTTTTT SEQ ID NO: 131 Imager CalibrationTTTTCTAAGTCATGTTGTTGAAGAATTTTTTT (Medium) SEQ ID NO: 126Cannabis ITS1 DNA TTTTTTAATCTGCGCCAAGGAACAATATTTTTTT Control 1SEQ ID NO: 127 Cannabis ITS1 DNA TTTTTGCAATCTGCGCCAAGGAACAATATTTTTTControl 2 SEQ ID NO: 128 Cannabis ITS1 DNATTTATTTCTTGCGCCAAGGAACAATATTTTATTT Control 3 SEQ ID NO: 86Total Yeast and Mold TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT(High sensitivity) SEQ ID NO: 87 Total Yeast and MoldTTTTTTTTGAATCATCGARTCTCCTTTTTTT (Low sensitivity) SEQ ID NO: 88Total Yeast and Mold TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT(Medium sensitivity) SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 92 Aspergillus fumigatus 1TTTCTTTTCGACACCCAACTTTATTTCCTTATTT SEQ ID NO: 90 Aspergillus flavus 1TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT SEQ ID NO: 95 Aspergillus niger 1TTTTTTCGACGTTTTCCAACCATTTCTTTT SEQ ID NO: 100 Botrytis spp.TTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTTTT SEQ ID NO: 108 Fusarium spp.TTTTTTTTAACACCTCGCRACTGGAGATTTTTTT SEQ ID NO: 89 Alternaria sppTTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT SEQ ID NO: 123 Rhodoturula spp.TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT SEQ ID NO: 117 Penicillium paxilliTTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT SEQ ID NO: 116 Penicillium oxalicumTTTTTTACACCATCAATCTTAACCAGGCCTTTTT SEQ ID NO: 118 Penicillium spp.TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT SEQ ID NO: 102 Candida spp. Group 1TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT SEQ ID NO: 103 Candida spp. Group 2TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT SEQ ID NO: 124 Stachybotrys sppTTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT SEQ ID NO: 125 Trichoderma spp.TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT SEQ ID NO: 105 Cladosporium spp.TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT SEQ ID NO: 121 Podosphaera spp.TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 37 Total Aerobic bacteriaTTTTTTTTTCCTACGGGAGGCAGTTTTTTT (High) SEQ ID NO: 38Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATTTTTTTT (Medium)SEQ ID NO: 39 Total Aerobic bacteria TTTATTTTCCCTACGGGAGGCTTTTATTTT(Low) SEQ ID NO: 47 Bile-tolerant Gram-TTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTTT negative (High) SEQ ID NO: 48Bile-tolerant Gram- TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT negative (Medium)SEQ ID NO: 49 Bile-tolerant Gram- TTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTTnegative (Low) SEQ ID NO: 53 Coliform/EnterobacteriaceaeTTTTTTCTATTGACGTTACCCGCTTTTTTT SEQ ID NO: 81 stx1 geneTTTTTTCTTTCCAGGTACAACAGCTTTTTT SEQ ID NO: 82 stx2 geneTTTTTTGCACTGTCTGAAACTGCCTTTTTT SEQ ID NO: 59 etuf geneTTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT SEQ ID NO: 65 Listeria spp.TTTTCTAAGTACTGTTGTTAGAGAATTTTT SEQ ID NO: 56 Aeromonas spp.TTATTTTCTGTGACGTTACTCGCTTTTATT SEQ ID NO: 78 Staphylococcus aureus 1TTTATTTTCATATGTGTAAGTAACTGTTTTATTT SEQ ID NO: 49 Campylobacter spp.TTTTTTATGACACTTTTCGGAGCTCTTTTT SEQ ID NO: 72 Pseudomonas spp.3TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT SEQ ID NO: 53 Clostridium spp.TTTTCTGGAMGATAATGACGGTACAGTTTT SEQ ID NO: 42 Escherichia coli/TTTTCTAATACCTTTGCTCATTGACTCTTT Shigella 1 SEQ ID NO: 74Salmonella enterica/ TTTTTTTGTTGTGGTTAATAACCGATTTTT Enterobacter 1SEQ ID NO: 61 invA gene TTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT

The set of 48 different probes of Table 4 were formulated as describedin Table 3, then printed onto epoxysilane coated borosilicate glass,using an Gentics Q-Array mini contact printer with Arrayit SMP pins,which deposit about 1 nL of formulation per spot. As described in FIG.1, the arrays thus printed were then allowed to react with theepoxisilane surface at room temperature, and then evaporate to removefree water, also at room temperature. Upon completion of the evaporationstep (typically overnight) the air-dried microarrays were then UVtreated in a Stratalinker UV irradiation system: 300 mjoules ofirradiation at 254 nm to initiate thymidine-mediated crosslinking. Themicroarrays are then ready for use, with no additional need for washingor capping.

Example 2

Using the 3-Dimensional Lattice Microarray System for DNA Analysis

Sample Processing

Harvesting Pathogens from plant surface comprises the following steps:

1. Wash the plant sample or tape pull in 1× phosphate buffered saline(PBS)

2. Remove plant material/tape

3. Centrifuge to pellet cells & discard supernatant

Resuspend in Sample Prep Buffer pre-mixed with Sample Digestion Buffer,sold under the trademark PathogenDx® and owned by PathogenDx, Inc.

5. Heat at 55° C. for 45 minutes

6. Vortex to dissipate the pellet

7. Heat at 95° C. for 15 minutes

8. Vortex and centrifuge briefly before use in PCR

Amplification by PCR

The sample used for amplification and hybridization analysis was aCannabis flower wash from a licensed Cannabis lab. The washed flowermaterial was then pelleted by centrifugation. The pellet was thendigested with proteinaseK, then spiked with a known amount of SalmonellaDNA before PCR amplification.

TABLE 5 PCR Primers and PCR conditions used in amplificationPCR primers (P1) for PCR Reaction #1 Cannabis ITS1 1°FP*-TTTGCAACAGCAGAACGACCCGTGA Cannabis ITS1 1°RP*-TTTCGATAAACACGCATCTCGATTG Enterobacteriaceae 16S 1°FP-TTACCTTCGGGCCTCTTGCCATCRGATGTG Enterobacteriaceae 16S 1°RP-TTGGAATTCTACCCCCCTCTACRAGACTCAAGCPCR primers (P2) for PCR Reaction #2 Cannabis ITS1 2°FP-TTTCGTGAACACGTTTTAAACAGCTTG Cannabis ITS1 2°RP-(Cy3)TTTTCCACCGCACGAGCCACGCGAT Enterobacteriaceae 16S 2°FP-TTATATTGCACAATGGGCGCAAGCCTGATG  Enterobacteriaceae 16S 2°RP-(Cy3)TTTTGTATTACCGCGGCTGCTGGCA Primary PCR Secondary PCR PCR ReagentConcentration Concentration PCR Buffer 1X 2X MgCl₂ 2.5 mM 2.5 mM BSA0.16 mg/mL 0.16 mg/mL dNTP's 200 mM 200 mM Prime mix 200 nM each50 nM-FP/200 Taq Polymerase 1.5 Units 1.5 UnitsProgram for PCR Reaction #1 95° C., 4 min 98° C., 30s 61° C., 30s 72°C., 60s 72° C., 7 min 25X Program for PCR Reaction #2 95° C., 4 min 98°C., 20s 61° C., 20s 72° C., 30s 72° C., 7 min 25X *FP, Forward Primer;*RP, Reverse Primer

The Salmonella DNA spiked sample was then amplified with PCR primers(P1—Table 5) specific for the 16S region of Enterobacteriaceae in atandem PCR reaction to first isolate the targeted region (PCR Reaction#1) and also PCR primers (P1—Table 5) which amplify a segment ofCannabis DNA (ITS) used as a positive control.

The product of PCR Reaction #1 (14) was then subjected to a second PCRreaction (PCR Reaction #2) which additionally amplified and labelled thetwo targeted regions (16S, ITS) with green CY3 fluorophore labeledprimers (P2—Table 5). The product of the PCR Reaction #2 (50 μL) wasthen diluted 1-1 with hybridization buffer (4×SSC+5×Denhardt's solution)and then applied directly to the microarray for hybridization.

Hybridization

Because the prior art method of microarray manufacture allows DNA to beanalyzed via hybridization without the need for pre-treatment of themicroarray

surface, the use of the microarray is simple, and involves 6 manual orautomated pipetting steps.

1) Pipette the amplified DNA+binding buffer onto the microarray

2) Incubate for 30 minutes to allow DNA binding to the microarray(typically at room temperature, RT)

3) Remove the DNA+binding buffer by pipetting

4) Pipette 50 uL of wash buffer onto the microarray(0.4×SSC+0.5×Denhardt's) and incubate 5 min at RT.

5) Remove the wash buffer by pipetting

6) Repeat steps 4&5

7) Perform image analysis at 532 nm and 635 nm to detect the probe spotlocation (532 nm) and PCR product hybridization (635 nm).

Image Analysis

Image Analysis was performed at two wavelengths (532 nm and 635 nm) on araster-based confocal scanner: GenePix 4000B Microarray Scanner, withthe following imaging conditions: 33% Laser power, 400 PMT setting at532 nm/33% Laser Power, 700 PMT setting at 635 nm. FIG. 3 shows anexample of the structure and hybridization performance of themicroarray.

FIG. 3A reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized at 635 nm. The 635nm image is derived from signals from the (red) CY5 fluor attached tothe 5′ terminus of the bifunctional polymer linker OligodT which hadbeen introduced during microarray fabrication as a positional marker ineach microarray spot (see FIG. 1 and Table 3). The data in FIG. 3Aconfirm that the Cy5-labelled OligodT has been permanently linked to themicroarray surface, via the combined activity of the bi-functionallinker and subsequent UV-crosslinking, as described in FIG. 1.

FIG. 3B reveals imaging of the representative microarray described aboveafter hybridization and washing as visualized at 532 nm. The 532 nmimage is derived from signals from the (green) CY3 fluor attached to the5′ terminus of PCR amplified DNA obtained during PCR Reaction #2. It isclear from FIG. 3B that only a small subset of the 48 discrete probesbind to the Cy3-labelled PCR product, thus confirming that the presentmethod of linking nucleic acid probes to form a microarray (FIG. 1)yields a microarray product capable of sequence specific binding to a(cognate) solution state target. The data in FIG. 3B reveal theunderlying 3-fold repeat of the data (i.e., the array is the same set of48 probes printed three times as 3 distinct sub-arrays to form the final48×3=144 element microarray. The observation that the same set of 48probes can be printed 3-times, as three repeated sub-domains show thatthe present invention generates microarray product that is reproducible.

FIG. 3C reveals imaging of the representative microarray, describedabove, after hybridization and washing, as visualized with both the 532nm and 635 nm images superimposed. The superimposed images display theutility of parallel attachment of a Cy5-labelled OligodT positionalmarker relative to the sequence specific binding of the CY3-labelled PCRproduct.

Example 3

FIG. 4A shows an exemplar of the first PCR step. As is standard, suchPCR reactions are initiated by the administration of PCR Primers.Primers define the start and stopping point of the PCR based DNAamplification reaction. In this embodiment, a pair of PCR reactions isutilized to support the needed DNA amplification. In general, such PCRamplification is performed in series: a first pair of PCRs, with thesuffix “P1” in FIG. 4A are used to amplify about 1 μL of any unpurifiedDNA sample, such as a raw Cannabis leaf wash for example. About 1 μL ofthe product of that first PCR reaction is used as the substrate for asecond PCR reaction that is used to affix a fluorescent dye label to theDNA, so that the label may be used to detect the PCR product when itbinds by hybridization to the microarray. The primer sequences for thefirst and second PCRs are shown in Table 6. The role of this two-stepreaction is to avert the need to purify the pathogen DNA to be analyzed.The first PCR reaction, with primers “P1” is optimized to accommodatethe raw starting material, while the second PCR primer pairs “P2” areoptimized to obtain maximal DNA yield, plus dye labeling from theproduct of the first reaction. Taken in the aggregate, the sum of thetwo reactions obviates the need to either purify or characterize thepathogen DNA of interest.

FIG. 4A reveals at low resolution the 16S rDNA region which is amplifiedin an embodiment, to isolate and amplify a region which may besubsequently interrogated by hybridization. The DNA sequence of this 16SrDNA region is known to vary greatly among different bacterial species.Consequently, having amplified this region by two step PCR, thatsequence variation may be interrogated by the subsequent microarrayhybridization step.

TABLE 6 First and Second PCR Primers SEQ ID NO. Primer targetPrimer sequence First PCR Primers (P1) for the first amplification stepSEQ ID NO: 1 16S rDNA HV3 Locus TTTCACAYTGGRACTGAGACACG (Bacteria)SEQ ID NO: 2 16S rDNA HV3 Locus TTTGACTACCAGGGTATCTAATCCTGT (Bacteria)SEQ ID NO: 3 Stx1 Locus TTTATAATCTACGGCTTATTGTTGAACG(Pathogenic E. coli) SEQ ID NO: 4 Stx1 LocusTTTGGTATAGCTACTGTCACCAGACAATG (Pathogenic E. coli) SEQ ID NO: 5Stx2 Locus TTTGATGCATCCAGAGCAGTTCTGCG (Pathogenic E. coli) SEQ ID NO: 6Stx2 Locus TTTGTGAGGTCCACGTCTCCCGGCGTC (Pathogenic E. coli) SEQ ID NO: 7InvA Locus (Salmonella) TTTATTATCGCCACGTTCGGGCAATTCG SEQ ID NO: 8InvA Locus (Salmonella) TTTCTTCATCGCACCGTCAAAGGAACCG SEQ ID NO: 9tuf Locus (All E. coli) TTTCAGAGTGGGAAGCGAAAATCCTG SEQ ID NO: 10tuf Locus (All E. coli) TTTACGCCAGTACAGGTAGACTTCTG SEQ ID NO: 1116S rDNA TTACCTTCGGGCCTCTTGCCATCRGATGTG Enterobacteriaceae HV3 LocusSEQ ID NO: 12 16S rDNA TTGGAATTCTACCCCCCTCTACRAGACTCAAGCEnterobacteriaceae HV3 Locus SEQ ID NO: 13 ITS2 Locus (All Yeast,TTTACTTTYAACAAYGGATCTCTTGG Mold/Fungus) SEQ ID NO: 14ITS2 Locus (All Yeast, TTTCTTTTCCTCCGCTTATTGATATG Mold/Fungus)SEQ ID NO: 15 ITS2 Locus TTTAAAGGCAGCGGCGGCACCGCGTCCG(Aspergillus species) SEQ ID NO: 16 ITS2 LocusTTTTCTTTTCCTCCGCTTATTGATATG (Aspergillus species) SEQ ID NO: 17ITS1 Locus TTTGCAACAGCAGAACGACCCGTGA (Cannabis/Plant) SEQ ID NO: 18ITS1 Locus TTTCGATAAACACGCATCTCGATTG (Cannabis/Plant)Second PCR Primers (P2) for the second labeling amplification stepSEQ ID NO: 19 16S rDNA HV3 Locus TTTACTGAGACACGGYCCARACTC (All Bacteria)SEQ ID NO: 20 16S rDNA HV3 Locus TTTGTATTACCGCGGCTGCTGGCA (All Bacteria)SEQ ID NO: 21 Stx1 Locus TTTATGTGACAGGATTTGTTAACAGGAC(Pathogenic E. coli) SEQ ID NO: 22 Stx1 LocusTTTCTGTCACCAGACAATGTAACCGCTG (Pathogenic E. coli) SEQ ID NO: 23Stx2 Locus TTTTGTCACTGTCACAGCAGAAG (Pathogenic E. coli) SEQ ID NO: 24Stx2 Locus TTTGCGTCATCGTATACACAGGAGC (Pathogenic E. coli) SEQ ID NO: 25InvA Locus (All Salmonella) TTTTATCGTTATTACCAAAGGTTCAG SEQ ID NO: 26InvA Locus (All Salmonella) TTTCCTTTCCAGTACGCTTCGCCGTTCG SEQ ID NO: 27tuf Locus (All E. coli) TTTGTTGTTACCGGTCGTGTAGAAC SEQ ID NO: 28tuf Locus (All E. coli) TTTCTTCTGAGTCTCTTTGATACCAACG SEQ ID NO: 2916S rDNA TTATATTGCACAATGGGCGCAAGCCTGATG Enterbacteriaceae HV3 LocusSEQ ID NO: 30 16S rDNA TTTTGTATTACCGCGGCTGCTGGCA Enterbacteriaceae HV3Locus SEQ ID NO: 31 ITS2 Locus (All Yeast, TTTGCATCGATGAAGARCGYAGCMold/Fungus) SEQ ID NO: 32 ITS2 Locus (All Yeast, TTTCCTCCGCTTATTGATATGCMold/Fungus) SEQ ID NO: 33 ITS2 Locus TTTCCTCGAGCGTATGGGGCTTTGTC(Aspergillus species) SEQ ID NO: 34 ITS2 Locus TITTTCCTCCGCTTATIGATATGC(Aspergillus species) SEQ ID NO: 35 ITS1 LocusTTTCGTGAACACGTTTTAAACAGCTTG (Cannabis/Plant) SEQ ID NO: 36 ITS1 LocusTTTCCACCGCACGAGCCACGCGAT (Cannabis/Plant)

FIG. 4B displays the stx1 gene locus which is present in the mostimportant pathogenic strains of E co/i and which encodes Shigatoxin 1.Employing the same two-step PCR approach, a set of two PCR primer pairswere designed which, in tandem, can be used to amplify and labelunprocessed bacterial samples to present the stx1 locus for analysis bymicroarray-based DNA hybridization.

FIG. 5A displays the stx2 gene locus which is also present in the mostimportant pathogenic strains of E coli and which encodes Shigatoxin 2.Employing the same two-step PCR approach, a set of two PCR primer pairswere designed which, in tandem, can be used to amplify and labelunprocessed bacterial samples so as to present the stx2 locus foranalysis by microarray-based DNA hybridization.

FIG. 5B displays the invA gene locus which is present in all strains ofSalmonella and which encodes the InvAsion A gene product. Employing thesame two-step PCR approach, a set of two PCR primer pairs were designedwhich, in tandem, can be used to amplify and label unprocessed bacterialsamples so as to present the invA locus for analysis by microarray-basedDNA hybridization.

FIG. 6 displays the tuf gene locus which is present in all strains of Ecoli and which encodes the ribosomal elongation factor Tu. Employing thesame two-step PCR approach, a set of two PCR primer pairs were designedwhich, in tandem, can be used to amplify and label unprocessed bacterialsamples so as to present the tuf locus for analysis by microarray-basedDNA hybridization.

FIG. 7 displays the ITS2 locus which is present in all eukaryotes,including all strains of yeast and mold and which encodes the intergenicregion between ribosomal genes 5.8S and 28S. ITS2 is highly variable insequence and that sequence variation can be used to resolve straindifferences in yeast, and mold. Employing the same two-step PCRapproach, a set of two PCR primer pairs were designed which, in tandem,can be used to amplify and label unprocessed yeast and mold samples soas to present the ITS2 locus for analysis by microarray-based DNAhybridization.

FIG. 8 displays the ITS1 gene locus which is present in all eukaryotes,including all plants and animals, which encodes the intergenic regionbetween ribosomal genes 18S and 5.8S. ITS1 is highly variable insequence among higher plants and that sequence variation can be used toidentify plant species. Employing the same two-step PCR approach, a setof two PCR primer pairs were designed which, in tandem, can be used toamplify and label unprocessed Cannabis samples so as to present the ITS1locus for analysis by microarray-based DNA hybridization. Theidentification and quantitation of the Cannabis sequence variant of ITS1is used as an internal normalization standard in the analysis ofpathogens recovered from the same Cannabis samples.

Table 7 displays representative oligonucleotide sequences which are usedas microarray probes in an embodiment for DNA microarray-based analysisof bacterial 16S locus as described in FIG. 4. The sequence of thoseprobes has been varied to accommodate the cognate sequence variationwhich occurs as a function of species difference among bacteria. In allcases, the probe sequences are terminated with a string of T's at eachend, to enhance the efficiency of probe attachment to the microarraysurface, at time of microarray manufacture. Table 8 shows sequences ofthe Calibration and Negative controls used in the microarray.

Table 9 displays representative oligonucleotide sequences which are usedas microarray probes in an embodiment for DNA microarray-based analysisof eukaryotic pathogens (fungi, yeast & mold) based on their ITS2 locusas described in FIG. 7. Sequences shown in Table 8 are used as controls.The sequence of those probes has been varied to accommodate the cognatesequence variation which occurs as a function of species differenceamong fungi, yeast & mold. In all cases, the probe sequences areterminated with a string of T's at each end, to enhance the efficiencyof probe attachment to the microarray surface, at time of microarraymanufacture.

Table 10 displays representative oligonucleotide sequences which areused as microarray probes in an embodiment for DNA microarray-basedanalysis of Cannabis at the ITS1 locus (Cannabis spp.).

TABLE 7 Oligonucleotide probe sequence for the 16S Locus SEQ ID NO: 37Total Aerobic bacteria TTTTTTTTTCCTACGGGAGGCAGTTTTTTT (High)SEQ ID NO: 38 Total Aerobic bacteria TTTTTTTTCCCTACGGGAGGCATTTTTTTT(Medium) SEQ ID NO: 39 Total Aerobic bacteria (Low)TTTATTTTCCCTACGGGAGGCTTTTATTTT SEQ ID NO: 40 EnterobacteriaceaeTTTATTCTATTGACGTTACCCATTTATTTT (Low sensitivity) SEQ ID NO: 41Enterobacteriaceae TTTTTTCTATTGACGTTACCCGTTTTTTTT (Medium sensitivity)SEQ ID NO: 42 Escherichia coli/Shigella 1 TTTTCTAATACCTTTGCTCATTGACTCTTTSEQ ID NO: 43 Escherichia coli/Shigella 2 TTTTTTAAGGGAGTAAAGTTAATATTTTTTSEQ ID NO: 44 Escherichia coli/Shigella 3 TTTTCTCCTTTGCTCATTGACGTTATTTTTSEQ ID NO: 45 Bacillus spp. Group1 TTTTTCAGTTGAATAAGCTGGCACTCTTTTSEQ ID NO: 46 Bacillus spp. Group2 TTTTTTCAAGTACCGTTCGAATAGTTTTTTSEQ ID NO: 47 Bile-tolerant Gram-negativeTTTTTCTATGCAGTCATGCTGTGTGTRTGTCTTTTT (High) SEQ ID NO: 48Bile-tolerant Gram-negative TTTTTCTATGCAGCCATGCTGTGTGTRTTTTTTT (Medium)SEQ ID NO: 49 Bile-tolerant Gram-negativeTTTTTCTATGCAGTCATGCTGCGTGTRTTTTTTT (Low) SEQ ID NO: 50Campylobacter spp. TTTTTTATGACACTTTTCGGAGCTCTTTTT SEQ ID NO: 51Chromobacterium spp. TTTTATTTTCCCGCTGGTTAATACCCTTTATTTT SEQ ID NO: 52Citrobacter spp. Group1 TTTTTTCCTTAGCCATTGACGTTATTTTTT SEQ ID NO: 53Clostridium spp. TTTTCTGGAMGATAATGACGGTACAGTTTT SEQ ID NO: 54Coliform/Enterobacteriaceae TTTTTTCTATTGACGTTACCCGCTTTTTTT SEQ ID NO: 55Aeromonas TTTTTGCCTAATACGTRTCAACTGCTTTTT salmonicida/hydrophiliaSEQ ID NO: 56 Aeromonas spp. TTATTTTCTGTGACGTTACTCGCTTTTATTSEQ ID NO: 57 Alkanindiges spp. TTTTTAGGCTACTGRTACTAATATCTTTTTSEQ ID NO: 58 Bacillus pumilus TTTATTTAAGTGCRAGAGTAACTGCTATTTTATTSEQ ID NO: 59 etuf gene TTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT SEQ ID NO: 60Hafnia spp. TTTTTTCTAACCGCAGTGATTGATCTTTTT SEQ ID NO: 61 invA geneTTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT SEQ ID NO: 62 Klebsiella oxytocaTTTTTTCTAACCTTATTCATTGATCTTTTT SEQ ID NO: 63 Klebsiella pneumoniaeTTTTTTCTAACCTTGGCGATTGATCTTTTT SEQ ID NO: 64 Legionella spp.TTTATTCTGATAGGTTAAGAGCTGATCTTTATTT SEQ ID NO: 65 Listeria spp.TTTTCTAAGTACTGTTGTTAGAGAATTTTT SEQ ID NO: 66 Panteoa agglomeransTTTTTTAACCCTGTCGATTGACGCCTTTTT SEQ ID NO: 67 Panteoa stewartiiTTTTTTAACCTCATCAATTGACGCCTTTTT SEQ ID NO: 68 Pseudomonas aeruginosaTTTTTGCAGTAAGTTAATACCTTGTCTTTT SEQ ID NO: 69 Pseudomonas cannabinaTTTTTTTACGTATCTGTTTTGACTCTTTTT SEQ ID NO: 70 Pseudomonas spp. 1TTTTTTGTTACCRACAGAATAAGCATTTTT SEQ ID NO: 71 Pseudomonas spp. 2TTTTTTAAGCACTTTAAGTTGGGATTTTTT SEQ ID NO: 72 Pseudomonas spp. 3TTTATTTTAAGCACTTTAAGTTGGGATTTTATTT SEQ ID NO: 73 Salmonella bongoriTTTTTTTAATAACCTTGTTGATTGTTTTTT SEQ ID NO: 74 SalmonellaTTTTTTTGTTGTGGTTAATAACCGATTTTT enterica/Enterobacter 1 SEQ ID NO: 75Salmonella TTTTTTTAACCGCAGCAATTGACTCTTTTT enterica/Enterobacter 2SEQ ID NO: 76 Salmonella TTTTTTCTGTTAATAACCGCAGCTTTTTTTenterica/Enterobacter 3 SEQ ID NO: 77 Serratia spp.TTTATTCTGTGAACTTAATACGTTCATTTTTATT SEQ ID NO: 78 Staphylococcus aureus 1TTTATTTTCATATGTGTAAGTAACTGTTTTATTT SEQ ID NO: 79 Staphylococcus aureus 2TTTTTTCATATGTGTAAGTAACTGTTTTTT SEQ ID NO: 80 Streptomyces spp.TTTTATTTTAAGAAGCGAGAGTGACTTTTATTTT SEQ ID NO: 81 stx1 geneTTTTTTCTTTCCAGGTACAACAGCTTTTTT SEQ ID NO: 82 stx2 geneTTTTTTGCACTGTCTGAAACTGCCTTTTTT SEQ ID NO: 83 Vibrio spp.TTTTTTGAAGGTGGTTAAGCTAATTTTTTT SEQ ID NO: 84 Xanthamonas spp.TTTTTTGTTAATACCCGATTGTTCTTTTTT SEQ ID NO: 85 Yersinia pestisTTTTTTTGAGTTTAATACGCTCAACTTTTT

TABLE 8 Calibration and Negative Controls SEQ ID NO: 129Imager Calibration TTTTCTATGTATCGATGTTGAGAAATTTTTTT (High)SEQ ID NO: 130 Imager Calibration TTTTCTAGATACTTGTGTAAGTGAATTTTTTT (Low)SEQ ID NO: 131 Imager Calibration TTTTCTAAGTCATGTTGTTGAAGAATTTTTTT(Medium) SEQ ID NO: 132 Negative controlTTTTTTCTACTACCTATGCTGATTCACTCTTTTT

TABLE 9 Oligonucleotide probe sequence for the ITS2 Locus SEQ ID NO: 86Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACGCATTTTTTT Mold (Highsensitivity) SEQ ID NO: 87 Total Yeast andTTTTTTTTGAATCATCGARTCTCCTTTTTTT Mold (Low sensitivity) SEQ ID NO: 88Total Yeast and TTTTTTTTGAATCATCGARTCTTTGAACGTTTTTTT Mold (Mediumsensitivity) SEQ ID NO: 89 Alternaria spp.TTTTTTCAAAGGTCTAGCATCCATTAAGTTTTTT SEQ ID NO: 90 Aspergillus flavus 1TTTTTTCGCAAATCAATCTTTTTCCAGTCTTTTT SEQ ID NO: 91 Aspergillus flavus 2TTTTTTTCTTGCCGAACGCAAATCAATCTTTTTTTTTTTT SEQ ID NO: 92 AspergillusTTTCTTTTCGACACCCAACTTTATTTCCTTATTT fumigatus 1 SEQ ID NO: 93 AspergillusTTTTTTTGCCAGCCGACACCCATTCTTTTT fumigatus 2 SEQ ID NO: 94Aspergillus nidulans TTTTTTGGCGTCTCCAACCTTACCCTTTTT SEQ ID NO: 95Aspergillus niger 1 TTTTTTCGACGTTTTCCAACCATTTCTTTT SEQ ID NO: 96Aspergillus niger 2 TTTTTTTTCGACGTTTTCCAACCATTTCTTTTTT SEQ ID NO: 97Aspergillus niger 3 TTTTTTTCGCCGACGTTTTCCAATTTTTTT SEQ ID NO: 98Aspergillus terreus TTTTTCGACGCATTTATTTGCAACCCTTTT SEQ ID NO: 99Blumeria TTTATTTGCCAAAAMTCCTTAATTGCTCTTTTTT SEQ ID NO: 100 Botrytis sppTTTTTTTCATCTCTCGTTACAGGTTCTCGGTTCTTTTTTT SEQ ID NO: 101 Candida albicansTTTTTTTTTGAAAGACGGTAGTGGTAAGTTTTTT SEQ ID NO: 102 Candida spp.TTTTTTTGTTTGGTGTTGAGCRATACGTATTTTT Group 1 SEQ ID NO: 103 Candida spp.TTTTACTGTTTGGTAATGAGTGATACTCTCATTTT Group 2 SEQ ID NO: 104Chaetomium spp. TTTCTTTTGGTTCCGGCCGTTAAACCATTTTTTT SEQ ID NO: 105Cladosporium spp TTTTTTTTGTGGAAACTATTCGCTAAAGTTTTTT SEQ ID NO: 106Erysiphe spp. TTTCTTTTTACGATTCTCGCGACAGAGTTTTTTT SEQ ID NO: 107Fusarium oxysporum TTTTTTTCTCGTTACTGGTAATCGTCGTTTTTTT SEQ ID NO: 108Fusarium spp TTTTTTTTAACACCTCGCRACTGGAGATTTTTTT SEQ ID NO: 109Golovinomyces TTTTTTCCGCTTGCCAATCAATCCATCTCTTTTT SEQ ID NO: 110Histoplasma TTTATTTTTGTCGAGTTCCGGTGCCCTTTTATTT capsulatum SEQ ID NO: 111Isaria spp. TTTATTTTTCCGCGGCGACCTCTGCTCTTTATTT SEQ ID NO: 112Monocillium spp. TTTCTTTTGAGCGACGACGGGCCCAATTTTCTTT SEQ ID NO: 113Mucor spp. TTTTCTCCAVVTGAGYACGCCTGTTTCTTTT SEQ ID NO: 114Myrothecium spp. TTTATTTTCGGTGGCCATGCCGTTAAATTTTATT SEQ ID NO: 115Oidiodendron spp. TTTTTTTGCGTAGTACATCTCTCGCTCATTTTTT SEQ ID NO: 116Penicillium TTTTTTACACCATCAATCTTAACCAGGCCTTTTT oxalicum SEQ ID NO: 117Penicillium paxilli TTTTTTCCCCTCAATCTTTAACCAGGCCTTTTTT SEQ ID NO: 118Penicillium spp TTTTTTCAACCCAAATTTTTATCCAGGCCTTTTT SEQ ID NO: 119Phoma/Epicoccum spp. TTTTTTTGCAGTACATCTCGCGCTTTGATTTTTT SEQ ID NO: 120Podosphaera spp TTTTTTGACCTGCCAAAACCCACATACCATTTTT SEQ ID NO: 121Podosphaera spp. TTTTTTTTAGTCAYGTATCTCGCGACAGTTTTTT SEQ ID NO: 122Pythium oligandrum TTTTATTTAAAGGAGACAACACCAATTTTTATTT SEQ ID NO: 123Rhodoturula spp TTTTTTCTCGTTCGTAATGCATTAGCACTTTTTT SEQ ID NO: 124Stachybotrys spp TTTCTTCTGCATCGGAGCTCAGCGCGTTTTATTT SEQ ID NO: 125Trichoderma spp TTTTTCCTCCTGCGCAGTAGTTTGCACATCTTTT

Table 11 displays representative oligonucleotide sequences which areused as microarray probes in an embodiment for DNA microarray-basedanalysis of bacterial pathogens (stx1, stx2, invA, tuf) and for DNAanalysis of the presence host Cannabis at the ITS1 locus (Cannabisspp.). It should be noted that this same approach, with modifications tothe ITS1 sequence, could be used to analyze the presence of other planthosts in such extracts.

TABLE 10 Oligonucleotide probe sequence for the Cannabis ITS1 LocusSEQ ID NO: 126 Cannabis ITS1 DNA TTTTTTAATCTGCGCCAAGGAACAATATTTTTTTControl 1 SEQ ID NO: 127 Cannabis ITS1 DNATTTTTGCAATCTGCGCCAAGGAACAATATTTTTT Control 2 SEQ ID NO: 128Cannabis ITS1 DNA TTTATTTCTTGCGCCAAGGAACAATATTTTATTT Control 3

TABLE 11 Representative Microarray Probe Design for the PresentInvention: Bacterial Toxins, ITS1 (Cannabis) SEQ ID NO: 81 stx/geneTTTTTTCTTTCCAGGTACAACAGCTTTTTT SEQ ID NO: 82 stx2 geneTTTTTTGCACTGTCTGAAACTGCCTTTTTT SEQ ID NO: 59 etuf geneTTTTTTCCATCAAAGTTGGTGAAGAATCTTTTTT SEQ ID NO: 61 invA geneTTTTTTTATTGATGCCGATTTGAAGGCCTTTTTT SEQ ID NO: 126 Cannabis ITS1 DNATTTTTTAATCTGCGCCAAGGAACAATATTTTTTT Control 1

FIG. 9 shows a flow diagram to describe how an embodiment is used toanalysis the bacterial pathogen or yeast and mold complement of aCannabis or related plant sample. Pathogen samples can be harvested fromCannabis plant material by tape pulling of surface bound pathogen or bysimple washing of the leaves or buds or stems, followed by a singlemultiplex “Loci Enhancement” Multiplex PCR reaction, which is thenfollowed by a single multiplex “Labelling PCR”. A different pair of twostep PCR reactions is used to analyze bacteria, than the pair of twostep PCR reactions used to analyze fungi, yeast & mold. In all cases,the DNA of the target bacteria or fungi, yeast & mold are PCR amplifiedwithout extraction or characterization of the DNA prior to two step PCR.Subsequent to the Loci Enhancement and Labelling PCR steps, theresulting PCR product is simply diluted into binding buffer and thenapplied to the microarray test. The subsequent microarray steps requiredfor analysis (hybridization and washing) are performed at lab ambienttemperature.

FIG. 10 provide images of a representative implementation of microarraysused in an embodiment. In this implementation, all nucleic acid probesrequired for bacterial analysis, along with Cannabis DNA controls(Tables 7 and 10) are fabricated into a single 144 element (12×12)microarray, along with additional bacterial and Cannabis probes such asthose in Table 10. In this implementation, all nucleic acid probesrequired for fungi, yeast & mold analysis along with Cannabis DNAcontrols were fabricated into a single 144 element (12×12) microarray,along with additional fungal probes shown in Table 9. The arrays aremanufactured on PTFE coated glass slides as two columns of 6 identicalmicroarrays. Each of the 12 identical microarrays is capable ofperforming, depending on the nucleic acid probes employed, a completemicroarray-based analysis bacterial analysis or a completemicroarray-based analysis of fungi, yeast & mold. Nucleic acid probeswere linked to the glass support via microfluidic printing, eitherpiezoelectric or contact based or an equivalent. The individualmicroarrays are fluidically isolated from the other 11 in this case, bythe hydrophobic PTFE coating, but other methods of physical isolationcan be employed.

FIGS. 11A-11B display representative DNA microarray analysis of anembodiment. In this case, purified bacterial DNA or purified fungal DNAhas been used, to test for affinity and specificity subsequent to thetwo-step PCR reaction and microarray-based hybridization analysis. Ascan be seen, the nucleic acid probes designed to detect each of thebacterial DNA (top) or fungal DNA (bottom) have bound to the target DNAcorrectly via hybridization and thus have correctly detected thebacterium or yeast. FIG. 12 displays representative DNA microarrayanalysis of an embodiment. In this case, 5 different unpurified rawCannabis leaf wash samples were used to test for affinity andspecificity subsequent to the two-step PCR reaction and microarray-basedhybridization analysis. Both bacterial and fungal analysis has beenperformed on all 5 leaf wash samples, by dividing each sample intohalves and subsequently processing them each for analysis of bacteria orfor analysis of fungi, yeast & mold. The data of FIG. 12 were obtainedby combining the outcome of both assays. FIG. 12 shows that thecombination of two step PCR and microarray hybridization analysis, asdescribed in FIG. 9, can be used to analyze the pathogen complement of aroutine Cannabis leaf wash. It is expected, but not shown that suchwashing of any plant material could be performed similarly.

FIG. 13 displays representative DNA microarray analysis of anembodiment. In this case, one unpurified (raw) Cannabis leaf wash samplewas used and was compared to data obtained from a commercially-obtainedhomogenous yeast vitroid culture of live Candida to test for affinityand specificity subsequent to the two-step PCR reaction andmicroarray-based hybridization analysis. Both Cannabis leaf wash andcultured fungal analysis have been performed by processing them each foranalysis via probes specific for fungi (see Tables 9 and 11).

The data of FIG. 13 were obtained by combining the outcome of analysisof both the leaf wash and yeast vitroid culture samples. The data ofFIG. 13 show that the combination of two step PCR and microarrayhybridization analysis, as described in FIG. 9, can be used tointerrogate the fungal complement of a routine Cannabis leaf wash asadequately as can be done with a pure (live) fungal sample. It isexpected that fungal analysis via such washing of any plant materialcould be performed similarly.

FIG. 14 shows a graphical representation of the position of PCR primersemployed in a variation of an embodiment for low level detection ofBacteria in the Family Enterobacteriaceae including E. coli. These PCRprimers are used to selectively amplify and dye label DNA from targetedorganisms for analysis via microarray hybridization.

FIGS. 15A-15C illustrate representative DNA microarray analysisdemonstrating assay sensitivity over a range of microbial inputs. Inthis case, certified reference material consisting of enumeratedbacterial colonies of E. coli O157:H7, E. coli O111 (FIGS. 15A, 15B) andSalmonella enterica (FIG. 15C) were spiked as a dilution series onto ahops plant surrogate matrix then processed using the assay versiondescribed for FIG. 14. Hybridization results from relevant probes fromFIG. 7 are shown. The larger numbers on the x-axis represents the totalnumber of bacterial colony forming units (CFU) that were spiked ontoeach hops plant sample, whereas the smaller numbers on the x-axisrepresent the number of CFU's of the spiked material that were actuallyinputted into the assay. Only about 1/50 of the original spiked hopssample volume was actually analyzed. The smaller numbers upon the x-axisof FIGS. 15A-15C are exactly 1/50^(th) that of the total (lower) values.As is seen, FIGS. 15A-15C show that the microarray test of an embodimentcan detect less than 1 CFU per microarray assay. The nucleic acidtargets within the bacterial genomes displayed in FIGS. 15A-15C comprise16S rDNA. There are multiple copies of the 16S rDNA gene in each ofthese bacterial organisms, which enables detection at <1 CFU levels.Since a colony forming unit approximates a single bacterium in manycases, the data of FIGS. 15A-15C demonstrate that the present microarrayassay has sensitivity which approaches the ability to detect a single(or a very small number) of bacteria per assay. Similar sensitivity isexpected for all bacteria and eukaryotic microbes in that it is knownthat they all present multiple copies of the ribosomal rDNA genes percell.

Tables 12A and 12B show a collection of representative microarrayhybridization data obtained from powdered dry food samples with noenrichment and 18-hour enrichment for comparison. The data shows thatbacterial microbes were successfully detected on the microarrays of thepresent invention without the need for enrichment.

FIG. 16 and Tables 13-15 describes embodiments for the analysis offruit, embodiments for the analysis of vegetables and embodiments forthe analysis of other plant matter. The above teaching shows, byexample, that unprocessed leaf and bud samples in Cannabis and hops maybe washed in an aqueous water solution, to yield a water-wash containingmicrobial pathogens which can then be analyzed via the presentcombination of RSG and microarrays.

If fresh leaf, flower, stem or root materials from fruit and vegetablesare also washed in a water solution in that same way (when fresh, orafter drying and grinding or other types or processing, then the presentcombination of RSG and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes in thoseother plant materials. At least two methods of sample collection arepossible for fruit and vegetables. One method is the simple rinsing ofthe fruit, exactly as described for Cannabis, above. Another method ofsample collection from fruits and vegetables is a “tape pull”, wherein apiece of standard forensic tape is applied to the surface of the fruit,then pulled off. Upon pulling, the tape is then soaked in the standardwash buffer described above, to suspend the microbes attached to thetape. Subsequent to the tape-wash step, all other aspects of theprocessing and analysis (i.e., raw sample genotyping, PCR, thenmicroarray analysis) are exactly as described above.

TABLE 12A Representative microarray data obtained from powdered dry foodsamples Sample Type Whey Protein Whey Protein Chewable Vanilla Pea ShakeVanilla Shake Chocolate Berry Tablet Shake Protein Enrichment time 0 180 18 0 18 0 18 0 18 hours hours hours hours hours hours hours hourshours hours Negative Control Probe 289 318 349 235 327 302 358 325 321299 Total Aerobic Bacteria Probes High sensitivity 26129 38896 1662911901 3686 230 32747 12147 41424 40380 Medium sensitivity 5428 6364 33082794 876 215 7310 2849 15499 8958 Low sensitivity 2044 3419 1471 990 446181 2704 1062 4789 3887 Bile-tolerant Gram-negative Probes Highsensitivity 2639 350 1488 584 307 305 1041 472 15451 8653 Mediumsensitivity 1713 328 892 493 322 362 615 380 6867 4997 Low sensitivity974 600 749 621 595 688 821 929 2459 1662 Total EnterobacteriaceaeProbes High sensitivity 1131 306 363 310 346 318 273 331 4260 3149Medium sensitivity 479 296 320 297 329 339 314 342 1489 990 Lowsensitivity 186 225 203 165 205 181 207 200 216 259 16S rDNA SpeciesProbes Escherichia coli/Shigella spp. 233 205 255 219 207 255 215 214242 198 S. enterica/enterobacter spp. 203 183 186 281 212 299 197 257308 303 Bacillus spp. 154 172 189 114 307 156 169 153 233 259Pseudomonas spp. 549 201 202 251 148 216 303 276 2066 983 OrganismSpecific Gene Probes tuf gene(E. coli) 204 129 180 272 158 190 191 183186 192 stx1 gene(E. coli) 241 178 171 240 289 304 195 245 149 191 stx2gene(E. coli) 145 96 136 125 182 224 130 142 85 127 invA (Salmonellaspp.) 215 265 210 284 204 256 239 285 237 229

TABLE 12B Representative microarray data obtained from powdered dry foodsamples Sample Type Rice Work-out Work-out Vanilla Protein Shake FPShake BR Shake Enrichment time 0 18 0 18 0 18 0 18 hours hours hourshours hours hours hours hours Negative Control Probe 351 351 271 309 299332 246 362 Total Aerobic Bacteria Probes High sensitivity 471 288 17146266 19207 261 41160 47198 Medium sensitivity 161 187 3120 229 3309 31110060 22103 Low sensitivity 186 239 1211 261 1223 264 3673 6750Bile-tolerant Gram-negative Probes High sensitivity 326 372 375 380 412363 1418 358 Medium sensitivity 304 362 341 391 308 356 699 394 Lowsensitivity 683 942 856 689 698 864 848 665 Total EnterobacteriaceaeProbes High sensitivity 277 329 317 327 298 326 290 349 Mediumsensitivity 326 272 296 291 297 263 262 307 Low sensitivity 215 207 204288 213 269 195 247 16S rDNA Species Probes Escherichia coli/Shigellaspp. 228 229 216 267 221 253 220 207 S. enterica/enterobacter spp. 226281 238 268 197 254 255 216 Bacillus spp. 157 166 812 208 915 216 415168 Pseudomonas spp. 199 225 247 251 211 259 277 225 Organism SpecificGene Probes tuf gene(E. coli) 150 149 126 206 163 212 215 166 stx1gene(E. coli) 270 247 211 299 239 307 175 185 stx2 gene(E. coli) 158 178127 205 138 175 128 100 invA (Salmonella spp.) 257 241 249 264 220 258239 245The data of Tables 13-15 demonstrates that simple washing of the fruitand tape pull sampling of the fruit generate similar microbial data. Theblueberry sample is shown to be positive for Botrytis, as expected,since Botrytis is a well-known fungal contaminant on blueberries. Thelemon sample is shown to be positive for Penicillium, as expected, sincePenicillium is a well-known fungal contaminant for lemons.

TABLE 13 Representative microarray hybridization data obtained fromblueberry and lemon washes. Sample Blueberry Lemon Produce Wash Wash 1blueberry in 2 ml Wash 1 piece moldy Collection Type 20 mM Borate,vortex 30 lemon in 2 ml 20 mM Protocol seconds Borate, vortex 30 secondsDilution Factor NONE 1:20 NONE 1:20 A. fumigatus 1 65 61 62 57 A.fumigatus 2 66 61 58 131 A. fumigatus 3 69 78 55 127 A. fumigatus 4 80198 63 161 A. fumigatus 5 98 68 59 70 A. flavus 1 111 65 197 58 A.flavus 2 64 66 71 49 A. flavus 3 72 79 54 49 A. flavus 4 95 71 66 125 A.flavus 5 59 55 45 47 A. niger 1 91 75 61 61 A. niger 2 185 68 61 57 A.niger 3 93 66 62 61 A. niger 4 1134 74 75 64 Botrytis spp. 1 26671 2760560 55 Botrytis spp. 2 26668 35611 59 57 Penicillium spp. 1 63 69 24444236 Penicillium spp. 2 71 69 4105 7426 Fusarium spp. 1 175 69 59 78Fusarium spp. 2 71 73 84 62 Mucor spp. 1 71 57 58 61 Mucor spp. 2 61 29066 61 Total Y & M 1 20052 21412 8734 7335 Total Y & M 2 17626 8454 55095030

TABLE 14 Representative microarray hybridization data obtained fromblueberry washes and tape pulls Sample Moldy Blueberry Collection TypeTape Pull ID 1A1 1A1 1A2 1A2 1A3 1A3 1B1 1B1 1B2 1B2 1B3 1B3 CollectionPoint 1 500 ul 20 mM Borate Buffer, vortex 30 seconds 500 ul 20 mMBorate + Triton Buffer, vortex 30 seconds Collection Point 2 Add 15 mgzirconia beads, Add 15 mg zirconia beads, vortex, Heat 5 min 95° C.,vortex, Heat 5 min 95° C., Vortex 15 seconds Vortex 15 secondsCollection Point 3 Heat 5 min Heat 5 min 95° C. vortex 95° C. vortex 15seconds 15 seconds Dilution Factor NONE 1:20 NONE 1:20 NONE 1:20 NONE1:20 NONE 1:20 NONE 1:20 A. fumigatus 1 66 388 83 77 97 313 95 68 76 5575 60 A. fumigatus 2 97 100 82 118 69 56 87 67 185 76 58 52 A. fumigatus3 77 94 82 1083 87 61 93 84 75 378 73 64 A. fumigatus 4 84 151 94 118 9680 115 85 85 93 190 88 A. fumigatus 5 63 75 96 71 78 61 98 74 68 98 70533 A. flavus 1 200 107 113 61 204 58 105 73 62 68 64 65 A. flavus 2 70104 64 57 133 281 111 78 377 314 57 50 A. flavus 3 83 90 94 150 99 90 96222 1162 86 80 73 A. flavus 4 76 125 92 146 87 174 241 78 115 69 105 85A. flavus 5 80 153 77 72 78 439 71 86 280 58 62 57 A. niger 1 409 178122 72 80 70 76 71 152 117 65 53 A. niger 2 78 292 79 65 715 666 74 7068 731 70 54 A. niger 3 86 76 87 558 78 60 70 81 96 63 478 58 A. niger 4164 70 92 108 197 69 130 75 76 148 73 65 Botrytis spp. 1 41904 2654928181 29354 25304 25685 57424 33783 57486 49803 33176 32153 Botrytisspp. 2 36275 25518 29222 27076 26678 27675 49480 32899 52817 34322 2969332026 Penicillium spp. 1 80 81 83 64 96 60 79 80 176 60 385 53Penicillium spp. 2 90 93 81 80 114 59 98 69 470 65 478 56 Fusarium spp.1 77 71 69 62 112 55 61 274 617 81 59 757 Fusarium spp. 2 91 82 107 74101 65 91 66 123 63 71 583 Mucor spp. 1 90 314 73 88 105 61 77 79 741180 172 74 Mucor spp. 2 83 69 73 69 91 67 111 102 455 88 70 133 Total Y& M 1 23637 18532 15213 17668 18068 19762 18784 15550 20625 17525 2581318269 Total Y & M 2 12410 8249 9281 11526 8543 13007 14180 14394 99058972 15112 12678The data embodied in FIG. 16 and Tables 13-15 demonstrate the use of anembodiment, for the recovery and analysis of yeast microbes on thesurface of fruit (blueberries and lemons in this case), but anembodiment could be extended to other fruits and vegetables for theanalysis of both bacterial and fungal contamination.

TABLE 15 Representative microarray hybridization data obtained fromlemon washes and tape pulls. Sample Moldy Lemon Collection Type TapePull ID 1A1 1A2 1A3 1B1 1B2 Lemon Lemon Lemon Lemon Lemon CollectionPoint 1 500 ul 20 mM Borate + Triton Buffer, vortex 30 secondsCollection Point 2 Add 15 mg Add 15 mg zirconia beads, zirconia beads,vortex, Heat 5 min vortex, Heat 5 min 95° C., Vortex 95° C., Vortex 15seconds 15 seconds Collection Point 3 Heat 5 min 95° C. vortex 15seconds Dilution Factor NONE A. fumigatus 1 96 83 75 83 64 A. fumigatus2 221 73 71 66 101 A. fumigatus 3 87 88 85 92 122 A. fumigatus 4 83 8591 72 97 A. fumigatus 5 448 100 84 114 78 A. flavus 1 85 79 70 66 63 A.flavus 2 77 82 77 79 63 A. flavus 3 133 66 86 60 67 A. flavus 4 96 85 8198 88 A. flavus 5 68 62 65 106 59 A. niger 1 73 88 77 73 73 A. niger 274 84 81 71 103 A. niger 3 90 86 87 74 78 A. niger 4 82 93 104 86 161Botrytis spp. 1 82 75 75 77 68 Botrytis spp. 2 91 74 83 67 62Penicillium spp. 1 3824 5461 5500 4582 5290 Penicillium spp. 2 7586 838011177 6528 8167 Fusarium spp. 1 101 62 61 70 279 Fusarium spp. 2 77 12278 68 233 Mucor spp. 1 74 110 89 76 57 Mucor spp. 2 132 1302 90 84 61Total Y & M 1 8448 12511 9249 12844 8593 Total Y & M 2 9275 8716 1158510758 4444

Table 16 shows embodiments for the analysis of environmental watersamples/specimens. The above teaching shows by example that unprocessedleaf and bud samples in Cannabis and hops may be washed in an aqueouswater solution, to yield a water-wash containing microbial pathogenswhich can then be analyzed via the present combination of Raw SampleGenotyping (RSG) and microarrays. If a water sample containing microbeswere obtained from environmental sources (such as well water, or seawater, or soil runoff or the water from a community water supply) andthen analyzed directly, or after ordinary water filtration toconcentrate the microbial complement onto the surface of the filter,that the present combination of RSG and microarray analysis would becapable of recovering and analyzing the DNA complement of thosemicrobes.

The data embodied in Table 16 were obtained from 5 well-water samples(named 2H, 9D, 21, 23, 25) along with 2 samples of milliQ laboratorywater (obtained via reverse osmosis) referred to as “Negative Control”.All samples were subjected to filtration on a sterile 0.4 um filter.Subsequent to filtration, the filters, with any microbial contaminationthat they may have captured, were then washed with the standard washsolution, exactly as described above for the washing of Cannabis andfruit. Subsequent to that washing, the suspended microbes in washsolution were then subjected to exactly the same combination ofcentrifugation (to yield a microbial pellet) then lysis and PCR of theunprocessed pellet-lysate (exactly as described above for Cannabis),followed by PCR and microarray analysis, also as described for Cannabis.

TABLE 16 Representative microarray data from raw water filtrate SampleID Negative 2 H 2 H 9 D 9 D 21 21 23 23 25 25 Control Imager CalibrationHigh 311 335 322 379 341 348 345 325 354 343 333 Imager Calibration Med280 314 268 286 288 231 253 295 267 295 244 Imager Calibration Low 245296 302 324 254 268 293 285 271 340 275 Cannabis cont. 310 330 313 255323 368 313 322 274 332 322 Cannabis cont. 313 237 298 271 298 288 296280 249 284 297 Cannabis cont. 208 265 276 250 267 327 255 258 253 282370 Total Yeast & Mold 284 324 290 307 272 361 296 288 271 321 469 TotalYeast & Mold 251 259 294 290 309 308 285 281 275 299 293 Total Yeast &Mold 282 280 294 280 299 284 275 286 299 259 232 Total Aerobic bacteriaHigh 40101 42007 47844 47680 45102 44041 43520 41901 46459 46783 135Total Aerobic bacteria Medium 14487 12314 24189 26158 19712 16210 1794315474 25524 18507 157 Total Aerobic bacteria Low 4885 5629 7625 64565807 4505 5316 6022 6264 6974 159 Negative Control 293 359 303 339 312329 306 377 307 335 307 Aspergillus fumigatus 285 291 284 268 289 265271 281 269 248 228 Aspergillus flavus 184 211 201 344 237 179 212 213163 204 171 Aspergillus niger 226 213 228 273 190 195 245 206 222 209172 Botrytis spp. 219 285 258 302 275 219 202 288 221 248 214 Alternariaspp. 81 97 76 89 58 76 75 175 117 174 167 Penicillium paxilli 135 162215 142 127 161 103 115 238 190 200 Penicillium oxalicum 119 107 161 131135 241 178 158 140 143 194 Penicillium spp. 50 123 179 177 128 138 146163 148 115 184 Can. alb/trop/dub 261 236 235 230 250 213 276 244 245237 194 Can. glab/Sach & Kluv spp. 146 165 196 128 160 215 185 217 215177 225 Podosphaera spp. 111 119 100 122 192 105 95 43 169 27 143Bile-tolerant Gram-negative High 16026 9203 13309 8426 16287 14116 1055717558 15343 14285 183 Bile-tolerant Gram-negative Medium 12302 119769259 10408 13055 10957 11242 8416 9322 11785 196 Bile-tolerantGram-negative Low 5210 7921 3818 3984 7224 6480 4817 6933 5021 5844 240Total Enterobacteriaceae High 193 248 389 357 215 214 198 220 276 208210 Total Enterobacteriaceae Med 246 214 297 246 244 224 219 245 252 229207 Total Enterobacteriaceae Low 165 140 158 119 151 180 150 167 182 174132 Total Coliform 121 148 158 117 129 117 155 157 125 178 152Escherichia coli specific gene 31821 115 132 155 127 62 86 121 59 90 234stx1 gene 67 0 2 0 0 23 21 28 0 0 116 stx2 gene 17 36 174 0 61 47 0 5133 0 85 Salmonella specific gene 181 172 245 172 178 212 157 243 174 156146 Bacillus spp. 137 135 174 112 164 143 163 182 168 152 149Pseudomonas spp. 271 74 332 56 366 133 91 114 60 179 555 Escherichiacoli/Shigella spp. 103 124 221 124 90 144 130 121 137 143 158 Salmonellaenterica/enterobacter spp. 124 98 131 119 136 88 121 77 128 140 124Erysiphe Group 2 278 221 237 230 245 254 250 220 205 236 233 Trichodermaspp. 105 157 204 152 180 154 130 161 201 180 150 Escherichia coli 429431 551 576 549 406 407 484 556 551 293 Aspergillus niger 218 212 216297 255 312 221 202 238 231 209 Escherichia coli/Shigella spp. 163 193220 202 308 280 121 271 341 317 124 Aspergillus fumigatus 713 865 862830 784 657 827 803 746 812 793 Aspergillus flavus 155 261 198 156 239171 250 218 210 258 219 Salmonella enterica 136 98 85 43 109 47 23 12370 100 135 Salmonella enterica 68 53 52 41 60 92 26 28 55 81 116

The data seen in Table 16 demonstrate that microbes collected onfiltrates of environmental water samples can be analyzed via the samecombination of raw sample genotyping, then PCR and microarray analysisused for Cannabis and fruit washes. The italicized elements of Table 16demonstrate that the 5 unprocessed well-water samples all containaerobic bacteria and bile tolerant gram-negative bacteria. The presenceof both classes of bacteria is expected for unprocessed (raw) wellwater. Thus, the data of Table 16 demonstrate that this embodiment ofthe present invention can be used for the analysis of environmentallyderived water samples.

The above teaching shows that unprocessed leaf and bud samples inCannabis and hops may be washed in an aqueous water solution to yield awater-wash containing microbial pathogens which can then be analyzed viathe present combination of RSG and microarrays. The above data also showthat environmentally-derived well water samples may be analyzed by anembodiment. Further, if a water sample containing microbes were obtainedfrom industrial processing sources (such as the water effluent from theprocessing of fruit, vegetables, grain, meat) and then analyzeddirectly, or after ordinary water filtration to concentrate themicrobial complement onto the surface of the filter, that the presentcombination of RSG and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes.

Further, if an air sample containing microbes as an aerosol or adsorbedto airborne dust were obtained by air filtration onto an ordinaryair-filter (such as used in the filtration of air in an agricultural orfood processing plant, or on factory floor, or in a public building or aprivate home) that such air-filters could then be washed with a watersolution, as has been demonstrated for plant matter, to yield amicrobe-containing filter eluate, such that the present combination ofRaw Sample Genotyping (RSG) and microarray analysis would be capable ofrecovering and analyzing the DNA complement of those microbes.

While the foregoing written description of an embodiments enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The presentdisclosure should therefore not be limited by the above describedembodiments, methods, and examples, but by all embodiments and methodswithin the scope and spirit of the present disclosure.

What is claimed is:
 1. A microarray system consisting of: asubstantially flat borosilicate glass slide with a front surfaceconsisting of: (1) activated surface moieties selected from the groupconsisting of an epoxysilane group, an N-hydroxysuccinimide group and anactivated carboxylic acid ester attached to the front surface; and (2) aplurality of oligodeoxythymidine linkers each covalently coupled at its3′ terminus via an amide bond to one of the activated surface moietygroups; wherein there are a greater number of activated surface moietiesattached to the front surface of the substantially flat borosilicateglass slide as compared to the total number of covalently coupledoligodeoxythymidine linkers in the plurality thereof, and wherein theactivated surface moieties that are not covalently coupled create alattice width spacing between the covalently coupled plurality ofoligodeoxythymidine linkers, each oligodeoxythymidine linker of saidplurality of oligodeoxythymidine linkers consisting of: (a) 20 to 60thymidine bases, with its 5′ terminus covalently linked to a fluorescentlabel; and (b) a plurality of nucleic acid probes selected from thegroup consisting of a plurality of pathogenic bacterial nucleotideprobes selected from the group consisting of SEQ ID NOS: 37-85, aplurality of pathogenic fungal nucleotide probes selected from the groupconsisting of SEQ ID NOS: 86-125, and a combination thereof, whereineach of said pathogenic bacterial nucleotide sequences probes of SEQ IDNOS: 37-85 and each of said pathogenic fungal nucleotide sequencesprobes of SEQ ID NOS: 86-126 consists of a pathogenic bacterialnucleotide sequence or a pathogenic fungal nucleotide sequencesandwiched between two to seven consecutive thymidine nucleotidesattached to both the 3′ terminus and to the 5′ terminus of eachpathogenic bacterial nucleotide sequence and each pathogenic fungalnucleotide sequence; wherein a thymidine nucleotide at the 3′ terminusand a thymidine nucleotide at the 5′ terminus of each of the pluralityof nucleic acid probes is crosslinked to two adjacentoligodeoxythymidine linkers of the plurality of oligodeoxythymidinelinkers attached to the substantially flat borosilicate glass slide; andwherein each of the plurality of nucleic acid probes crosslinked at the3′ terminus and the 5′ terminus to two adjacent oligodeoxythymidinelinkers that are covalently coupled to the substantially flatborosilicate glass slide are separated by both a vertical space and thelattice width, such that they form a 3-dimensional lattice on thesubstantially flat borosilicate glass slide.